Upregulation of type III endothelial cell nitric oxide synthase by agents that disrupt actin cytoskeletal organization

ABSTRACT

A use for agents that disrupt actin cytoskeletal organization is provided. In the instant invention, agents that disrupt actin cytoskeletal organization are found to upregulate endothelial cell Nitric Oxide Synthase activity. As a result, agents that disrupt actin cytoskeletal organization are useful in treating or preventing conditions that result from the abnormally low expression and/or activity of endothelial cell Nitric Oxide Synthase. Such conditions include hypoxia-induced conditions. Subjects thought to benefit mostly from such treatments include nonhyperlipidemics and nonhypercholesterolemics, but not necessarily exclude hyperlipidemics and hypercholesterolemics.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/115,387 filed on Jul. 14, 1998, entitled UPREGULATION OF TYPE IIIENDOTHELIAL CELL NITRIC OXIDE SYNTHASE BY AGENTS THAT DISRUPT ACTINCYTOSKELETAL ORGANIZATION. The contents of the above-identifiedapplications are hereby expressly incorporated by reference.

GOVERNMENT SUPPORT

[0002] The work resulting in this invention was supported in part by NIHGrant No. RO1-HL-52233. The U.S. Government may therefore be entitled tocertain rights in the invention

FIELD OF THE INVENTION

[0003] This invention relates to the use of agents that disrupt actincytoskeletal organization as upregulators of Type III endothelial cellNitric Oxide Synthase. Further, this invention relates to methods thatemploy agents that disrupt actin cytoskeletal organization to treatconditions that result from the abnormally low expression and/oractivity of endothelial cell Nitric Oxide Synthase in a subject.

BACKGROUND OF THE INVENTION

[0004] Nitric oxide (NO) has been recognized as an unusual messengermolecule with many physiologic roles, in the cardiovascular, neurologicand immune systems (Griffith, TM et al., J Am Coll Cardiol, 1988,12:797-806). It mediates blood vessel relaxation, neurotransmission andpathogen suppression. NO is produced from the guanidino nitrogen ofL-arginine by NO Synthase (Moncada, S and Higgs, E A, Eur J Clin Invest,1991, 21(4):361-374). In mammals, at least three isoenzymes of NOSynthase have been identified. Two, expressed in neurons (nNOS) andendothelial cells (Type III-ecNOS), are calcium-dependent, whereas thethird is calcium-independent and is expressed by macrophages and othercells after induction with cytokines (Type I-iNOS) (Bredt, D S andSnyder, S H, Proc Natl Acad Sci USA, 1990, 87:682-685, Janssens, S P etal., J Biol Chem, 1992, 267:22964, Lyons, C R et al., J Biol Chem, 1992,267:6370-6374). The various physiological and pathological effects of NOcan be explained by its reactivity and different routes of formation andmetabolism.

[0005] Recent studies suggest that a loss of endothelial-derived NOactivity may contribute to the atherogenic process (O'Driscoll, G, etal., Circulation, 1997, 95:1126-1131). For example, endothelial-derivedNO inhibits several components of the atherogenic process includingmonocyte adhesion to the endothelial surface (Tsao, PS et al.,Circulation, 1994, 89:2176-2182), platelet aggregation (Radomski, M W,et al., Proc Natl Acad Sci USA, 1990, 87:5193-5197), vascular smoothmuscle cell proliferation (Garg, U C and Hassid, A, J Clin Invest, 1989,83:1774-1777), and vasoconstriction (Tanner, FC et al., Circulation,1991, 83:2012-2020). In addition, NO can prevent oxidative modificationof low-density lipoprotein (LDL) which is a major contributor toatherosclerosis, particularly in its oxidized form (Cox, D A and Cohen,M L, Pharm Rev, 1996, 48:3-19).

[0006] It has been shown in the prior art that hypoxia downregulatesecNOS expression and/or activity via decreases in both ecNOS genetranscription and mRNA stability (Liao, J K et al., J Clin Invest, 1995,96:2661-2666, Shaul, P W et al., Am J Physiol, 1997, 272: L1005-L1012).Thus, ischemia-induced hypoxia may produce deleterious effects, in part,through decreases in ecNOS activity.

[0007] HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is themicrosomal enzyme that catalyzes the rate limiting reaction incholesterol biosynthesis (HMG-CoA6Mevalonate). An HMG-CoA reductaseinhibitor inhibits HMG-CoA reductase, and as a result inhibits thesynthesis of cholesterol. A number of HMG-CoA reductase inhibitors hasbeen used to treat individuals with hypercholesterolemia. Clinicaltrials with such compounds have shown great reductions of cholesterollevels in hypercholesterolemic patients. Moreover, it has been shownthat a reduction in serum cholesterol levels is correlated with improvedendothelium-dependent relaxations in atherosclerotic vessels (Treasure,CB et al., N Engl J Med, 1995, 332:481-487). Indeed, one of the earliestrecognizable benefits after treatment with HMG-CoA reductase inhibitorsis the restoration of endothelium-dependent relaxations or ecNOSactivity (supra, Anderson, T J et al., N Engl J Med, 1995, 332:488-493).

[0008] Although the mechanism by which HMG-CoA reductase inhibitorsrestore endothelial function is primarily attributed to the inhibitionof hepatic HMG-CoA reductase and the subsequent lowering of serumcholesterol levels, little is known on whether inhibition of endothelialHMG-CoA reductase has additional beneficial effects on endothelialfunction.

[0009] By inhibiting L-mevalonate synthesis, HMG-CoA reductaseinhibitors also prevent the synthesis of other important isoprenoidintermediates of the cholesterol biosynthetic pathway, such asfarnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP)(Goldstein, J L and Brown, M S, Nature, 1990, 343:425-430). Theisoprenoids are important lipid attachments for the post-translationalmodification of variety of proteins, including G-protein and G-proteinsubunits, Heme-a, nuclear lamins, Ras, and Ras-like proteins, such asRho, Rab, Rac, Ral or Rap (Goldstein, JL and Brown, MS, supra; Casey, PJ, Science, 1995, 268:221-225). The role that isoprenoids play inregulating ecNOS expression, however, is not known.

[0010] Pulmonary hypertension is a major cause of morbidity andmortality in individuals exposed to hypoxic conditions (Scherrer, U etal., N Engl J Med, 1996, 334:624-629). Recent studies demonstrate thatpulmonary arterial vessels from patients with pulmonary hypertensionhave impaired release of NO (Giaid, A and Saleh, D, N Engl J Med, 1995,333:214-221, Shaul, P W, Am J Physiol, 1997, 272: L1005-L1012).Additionally, individuals with pulmonary hypertension demonstratereduced levels of ecNOS expression in their pulmonary vessels andbenefit clinically from inhalation nitric oxide therapy (Roberts, J D etal., N Engl J Med, 1997, 336:605-610, Kouyoumdjian, C et al., J ClinInvest, 1994, 94:578-584). Conversely, mutant mice lacking ecNOS gene ornewborn lambs treated with the ecNOS inhibitor, Nw-monomethyl-L-arginine(LNMA), develop progressive elevation of pulmonary arterial pressuresand resistance (Steudel, W et al., Circ Res, 1997, 81:34-41, Fineman, JR et al., J Clin Invest, 1994, 93:2675-2683). It has also been shown inthe prior art that hypoxia causes pulmonary vasoconstriction viainhibition of endothelial cell nitric oxide synthase (ecNOS) expressionand activity (Adnot, S et al., J Clin Invest, 1991, 87:155-162, Liao, JK et al., J Clin Invest, 1995, 96, 2661-2666). Hence, hypoxia-mediateddownregulation of ecNOS may lead to the vasoconstrictive and structuralchanges associated with pulmonary hypertension.

[0011] Often cited as the third most frequent cause of death in thedeveloped countries, stroke has been defined as the abrupt impairment ofbrain function caused by a variety of pathologic changes involving oneor several intracranial or extracranial blood vessels. Approximately 80%of all strokes are ischemic strokes, resulting from restricted bloodflow. Mutant mice lacking the gene for ecNOS are hypertensive (Huang, PL et al., Nature, 1995, 377:239-242, Steudel, W et al., Circ Res, 1997,81:34-41) and develop greater intimal smooth muscle proliferation inresponse to cuff injury. Furthermore, occlusion of the middle cerebralartery results in 21% greater infarct size in “ecNOS knockout” micecompared to wildtype mice (Huang, Z et al., J Cereb Blood Flow Metab,1996, 16:981-987). These findings suggest that the ecNOS production mayplay a role in cerebral infarct formation and sizes. Additionally, sincemost patients with ischemic strokes have average or normal cholesterollevels, little is known on what the potential benefits of HMG-CoAreductase inhibitor administration would be in cerebrovascular events.

[0012] There exists a need to identify agents that improve endothelialcell function.

[0013] There also exists a need to identify agents that can be usedacutely or in a prophylactic manner to treat conditions that result fromlow levels of endothelial cell Nitric Oxide Synthase.

SUMMARY OF THE INVENTION

[0014] The invention involves the discovery that agents which disruptactin cytoskeletal organization can upregulate endothelial cell NitricOxide Synthase (Type III) expression. The invention, therefore, isuseful whenever it is desirable to restore endothelial cell Nitric OxideSynthase activity or increase such activity in a cell, tissue orsubject, provided the cell or the tissue expresses endothelial cellNitric Oxide Synthase.

[0015] Nitric Oxide Synthase activity is involved in many conditions,including impotence, heart failure, gastric and esophageal motilitydisorders, kidney disorders such as kidney hypertension and progressiverenal disease, insulin deficiency, etc. Individuals with such conditionswould benefit from increased endothelial cell Nitric Oxide Synthaseactivity. It also was known that individuals with pulmonary hypertensiondemonstrate reduced levels of Nitric Oxide Synthase expression in theirpulmonary vessels and benefit clinically from inhalation of NitricOxide. The invention therefore is particularly useful for treatingpulmonary hypertension. It also has been demonstrated that hypoxiacauses an inhibition of endothelial cell Nitric Oxide Synthase activity.The invention therefore is useful for treating subjects withhypoxia-induced conditions. It also has been discovered, surprisingly,that agents which disrupt actin cytoskeletal organization are useful forreducing brain injury that occurs following a stroke.

[0016] According to one aspect of the invention, a method is providedfor increasing endothelial cell Nitric Oxide Synthase activity in asubject who would benefit from increased endothelial cell Nitric OxideSynthase activity in a tissue. The method involves administering to asubject in need of such treatment an agent that disrupts actincytoskeletal organization in an amount(s) effective to increaseendothelial cell Nitric Oxide Synthase activity in the tissue of thesubject, provided that the agent that disrupts actin cytoskeletalorganization is not a rho GTPase function inhibitor. In one importantembodiment agents that disrupt actin cytoskeletal organization do notaffect cholesterol levels in a subject. In certain embodiments, however,agents that disrupt actin cytoskeletal organization as well asincreasing endothelial cell Nitric Oxide Synthase activity in the tissueof a subject can also affect cholesterol levels in the subject. Incertain embodiments, the subject is nonhyperlipidimic. In otherembodiments the amount is sufficient to increase endothelial cell NitricOxide Synthase activity above normal baseline levels established byage-controlled groups, described in greater detail below.

[0017] The subject can have a condition characterized by an abnormallylow level of endothelial cell Nitric Oxide Synthase activity which ishypoxia-induced. In other embodiments the subject can have a conditioncomprising an abnormally low level of endothelial cell Nitric OxideSynthase activity which is chemically induced. In still otherembodiments the subject can have a condition comprising an abnormallylow level of endothelial cell Nitric Oxide Synthase activity which iscytokine induced. In certain important embodiments, the subject haspulmonary hypertension or an abnormally elevated risk of pulmonaryhypertension. In other important embodiments, the subject hasexperienced an ischemic stroke or has an abnormally elevated risk of anischemic stroke. In still other important embodiments, the subject hasheart failure or progressive renal disease. In yet other importantembodiments, the subject is chronically exposed to hypoxic conditions.

[0018] According to any of the foregoing embodiments, the preferredagent that disrupts actin cytoskeletal organization is selected from thegroup consisting of a myosin light chain kinase inhibitor, a myosinlight chain phosphatase, a protein kinase N inhibitor, aphospatidylinositol 4-phosphate 5-kinase inhibitor, and cytochalasin D.In some embodiments the myosin light chain kinase inhibitor is selectedfrom the group consisting of 2,3-butanedione 2-monoxime,1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride, and 1-(5-isoquinolinesulphonyl) -2-methylpiperazinedihydro-chloride. Likewise, in any of the foregoing embodiments, themethod can further comprise co-administering an endothelial cell NitricOxide Synthase substrate and/or co-administering an agent other than anagent that disrupts actin cytoskeletal organization that also increasesendothelial cell Nitric Oxide Synthase activity, and/or co-administeringat least one different agent that disrupts actin cytoskeletalorganization. A preferred agent other than an agent that disrupts actincytoskeletal organization is selected from the group consisting ofestrogens and angiotensin-converting enzyme (ACE) inhibitors. The agentsmay be administered to a subject who has a condition or prophylacticallyto a subject who has a risk, and more preferably, an abnormally elevatedrisk, of developing a condition. The inhibitors also may be administeredacutely.

[0019] According to another aspect of the invention, a method isprovided for increasing endothelial cell Nitric Oxide Synthase activityin a subject to treat a condition favorably affected by an increase inendothelial cell Nitric Oxide Synthase activity in a tissue. Suchconditions are exemplified above. The method involves administering to asubject in need of such treatment an agent that disrupts actincytoskeletal organization in an amount effective to increase endothelialcell Nitric Oxide Synthase activity in the tissue of the subject,provided that the agent that disrupts actin cytoskeletal organization isnot a rho GTPase function inhibitor. In important embodiments, agentsthat disrupt actin cytoskeletal organization do not affect cholesterollevels in a subject. In certain embodiments, however, agents thatdisrupt actin cytoskeletal organization as well as increase endothelialcell Nitric Oxide Synthase activity in the tissue of a subject can alsoaffect cholesterol levels in the subject. In certain embodiments, thesubject is nonhyperlipidimic. Important conditions are as describedabove. Also as described above, the method can involve co-administrationof substrates of endothelial cell Nitric Oxide Synthase and/orco-administering an agent other than an agent that disrupts actincytoskeletal organization that also increases endothelial cell NitricOxide Synthase activity, and/or co-administering at least one differentagent that disrupts actin cytoskeletal organization. Preferred compoundsare as described above. As above, the agents that disrupt actincytoskeletal organization with or without the co-administered compoundscan be administered, inter alia, acutely or prophylactically.

[0020] According to another aspect of the invention, a method isprovided for reducing brain injury resulting from stroke. The methodinvolves administering to a subject having an abnormally high risk of anischemic stroke an agent that disrupts actin cytoskeletal organizationin an amount effective to increase endothelial cell Nitric OxideSynthase activity in the brain of the subject, provided that the agentthat disrupts actin cytoskeletal organization is not a rho GTPasefunction inhibitor. As above, important embodiments include the agentbeing selected from the group consisting of a myosin light chain kinaseinhibitor, a myosin light chain phosphatase, a protein kinase Ninhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, andcytochalasin D. As above, in some embodiments a myosin light chainkinase inhibitor is selected from the group consisting of2,3-butanedione 2-monoxime,1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride, and 1-(5-isoquinolinesulphonyl) -2-methylpiperazinedihydro-chloride. Also as above, important embodiments includeco-administering a substrate of endothelial cell Nitric Oxide Synthaseand/or co-administering an agent other than an agent that disrupts actincytoskeletal organization that also increases endothelial cell NitricOxide Synthase activity, and/or co-administering at least one differentagent that disrupts actin cytoskeletal organization. Likewise, importantembodiments include prophylactic and acute administration of theagent(s).

[0021] According to another aspect of the invention, a method isprovided for treating pulmonary hypertension. The method involvesadministering to a subject in need of such treatment an agent thatdisrupts actin cytoskeletal organization in an amount effective toincrease pulmonary endothelial cell Nitric Oxide Synthase activity inthe subject, provided that the agent that disrupts actin cytoskeletalorganization is not a rho GTPase function inhibitor. Particularlyimportant embodiments are as described above in connection with themethods for treating brain injury. Another important embodiment isadministering the agent prophylactically to a subject who has anabnormally elevated risk of developing pulmonary hypertension, includingsubjects that are chronically exposed to hypoxic conditions.

[0022] According to another aspect of the invention, a method fortreating heart failure is provided. The method involves administering toa subject in need of such treatment an agent that disrupts actincytoskeletal organization in an amount effective to increase vascularendothelial cell Nitric Oxide Synthase activity in the subject, providedthat the agent that disrupts actin cytoskeletal organization is not arho GTPase function inhibitor. As discussed above, important embodimentsinclude prophylactic and acute administration of the agent(s). Preferredcompounds and co-administration schemes are as described above.

[0023] According to yet another aspect of the invention, a method isprovided for treating progressive renal disease. The method involvesadministering to a subject in need of such treatment an agent thatdisrupts actin cytoskeletal organization in an amount effective toincrease renal endothelial cell Nitric Oxide Synthase activity in thekidney of the subject, provided that the agent that disrupts actincytoskeletal organization is not a rho GTPase function inhibitor.Important embodiments and preferred compounds and schemes ofco-administration are as described above in connection with heartfailure.

[0024] According to another aspect of the invention, a method forincreasing blood flow in a tissue of a subject is provided. The methodinvolves administering to a subject in need of such treatment a firstagent that disrupts actin cytoskeletal organization in an amounteffective to increase endothelial cell Nitric Oxide Synthase activity inthe tissue of the subject, provided that the first agent is not an agentselected from the group consisting of a rho GTPase function inhibitorand fasudil. In certain embodiments the first agent is not a myosinlight chain kinase inhibitor. In other embodiments the first agent isselected from the group consisting of a myosin light chain phosphatase,a protein kinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinaseinhibitor, and cytochalasin D. Other important embodiments includeco-administering a second agent to the subject with a conditiontreatable by the second agent in an amount effective to treat thecondition, whereby the delivery of the second agent to a tissue of thesubject is enhanced as a result of the increased blood flow. In certainembodiments where a second agent is administered, the conditiontreatable by the second agent does not involve the brain tissue.

[0025] The invention also involves the use of agents that disrupt actincytoskeletal organization in the manufacture of medicaments for treatingthe above-noted conditions. Important conditions, compounds, etc. are asdescribed above. The invention further involves pharmaceuticalpreparations that are cocktails of agents that disrupt actincytoskeletal organization according to the invention [non-rho GTPasefunction inhibitor(s)]. In certain embodiments, however, the cocktailscan include a rho GTPase function inhibitor(s) that disrupts actincytoskeletal organization together with the non-rho GTPase functioninhibitor agent of the invention. The invention also involvespharmaceutical preparations that are cocktails of agents that disruptactin cytoskeletal organization together with agents other than agentsthat disrupt actin cytoskeletal organization that also increase ecNOSactivity in a cell.

[0026] The invention also involves methods for increasing ecNOS activityin a cell by contacting the cell with an effective amount of an agentthat disrupts actin cytoskeletal organization (excluding rho GTPasefunction inhibitors), alone, or together with any of the agentsco-administered as described above, or as a cocktail as described above.

[0027] In any of the foregoing aspects of the invention the agent can bea non-fasudil agent that disrupts actin cytoskeletal organization.

[0028] These and other aspects of the invention are described in greaterdetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1. ecNOS activity and expression in wild-type SV-129 miceaortas with and without treatment with simvastatin for 14 days.

[0030]FIG. 2. ecNOS mRNA expression in the infarcted, ipsolateral (I)and not-infarcted, contralateral (C) forebrain hemispheres of SV-129mice with and without treatment with simvastatin.

[0031]FIG. 3. Northern blots showing the effects of mevastatin alone orin combination with FPP or GGPP on eNOS (ecNOS) steady-state mRNA levelsafter 24h.

[0032]FIG. 4. Western blots showing the effects of mevastatin alone orin combination with FPP or GGPP or LDL-cholesterol on eNOS (ecNOS)protein levels after 24h.

[0033]FIG. 5. Western blots showing the effects of C3 transferase,mevastatin, or L-mevalonate on eNOS (ecNOS) protein levels after 24h.

[0034]FIG. 6. Western blots showing eNOS (ecNOS) protein levels aftertransfection with insertless vector, pcDNA3 (C), c-myc-wildtype-RhoA(wt), and c-myc-N19RhoA (dominant-negative rhoA mutant).

[0035]FIG. 7. Effects of C3 transferase, FPP, GGPP, and CNF-1 onmevastatin-induced eNOS (ecNOS) activity as determined byLNMA-inhibitable nitrite production at 24 h.

[0036]FIG. 8. Immunoblots showing the concentration-dependent effects ofMLC kinase inhibitor H-7on ecNOS protein levels after 24 hours.

[0037]FIG. 9. Northern blots showing ecNOS expression of endothelialcells treated with cytochalasin D at 24 hours.

[0038]FIG. 10. Immunoblots showing the concentration-dependent effectsof 2, 3-butanedione 2-monoxime on ecNOS protein levels.

[0039]FIG. 11. Northern blots showing ecNOS expression of endothelialcells treated with nocodazole for 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The invention is useful whenever it is desirable to increaseendothelial cell Nitric Oxide Synthase (Type III isoform) activity in acell, in a tissue, or in a subject. A subject as used herein includeshumans, non human primates, dogs, cats, sheep, goats, cows, pigs, horsesand rodents. The invention thus is useful for therapeutic purposes andalso is useful for research purposes such as in testing in animal or invitro models of medical, physiological or metabolic pathways orconditions. Nitric Oxide Synthase is the enzyme that catalyzes thereaction that produces nitric oxide from the substrate L-arginine. Asthe name implies, endothelial cell nitric oxide Synthase refers to theType III isoform of the enzyme found in the endothelium.

[0041] By “ecNOS activity”, it is meant the ability of a cell togenerate nitric oxide from the substrate L-arginine. Increased ecNOSactivity can be accomplished in a number of different ways. For example,an increase in the amount of ecNOS protein or an increase in theactivity of the protein (while maintaining a constant level of theprotein) can result in increased “activity”. An increase in the amountof protein available can result from increased transcription of theecNOS gene, increased stability of the ecNOS mRNA or a decrease in ecNOSprotein degradation. (The term “expression” is used interchangeably withthe term “activity” throughout this application).

[0042] The ecNOS activity in a cell or in a tissue can be measured in avariety of different ways. A direct measure would be to measure theamount of ecNOS present. Another direct measure would be to measure theamount of conversion of arginine to citrulline by ecNOS or the amount ofgeneration of nitric oxide by ecNOS under particular conditions, such asthe physiologic conditions of the tissue. The ecNOS activity also can bemeasured more indirectly, for example by measuring mRNA half-life (anupstream indicator) or by a phenotypic response to the presence ofnitric oxide (a downstream indicator). One phenotypic measurementemployed in the art is detecting endothelial dependent relaxation inresponse to a acetylcholine, which response is affected by ecNOSactivity. The level of nitric oxide present in a sample can be measuredusing a nitric oxide meter. All of the foregoing techniques are wellknown to those of ordinary skill in the art, and some are described inthe examples below.

[0043] The present invention, by causing an increase in ecNOS activity,permits not only the re-establishment of normal base-line levels ofecNOS activity, but also allows increasing such activity above normalbase-line levels. Normal base-line levels are the amounts of activity ina normal control group, controlled for age and having no symptoms whichwould indicate alteration of endothelial cell Nitric Oxide Synthaseactivity (such as hypoxic conditions, hyperlipidemia and the like). Theactual level then will depend upon the particular age group selected andthe particular measure employed to assay activity. Specific examples ofvarious measures are provided below. In abnormal circumstances, e.g.hypoxic conditions, pulmonary hypertension, etc., endothelial cellNitric Oxide Synthase activity is depressed below normal levels.Surprisingly, when using agents that disrupt actin cytoskeletalorganization according to the invention, not only can normal base-linelevels be restored in such abnormal conditions, but endothelial cellNitric Oxide Synthase activity can be increased desirably far abovenormal base-line levels of endothelial cell Nitric Oxide Synthaseactivity. Thus, “increasing activity” means any increase in endothelialcell Nitric Oxide Synthase activity in the subject resulting from thetreatment with agents that disrupt actin cytoskeletal organizationaccording to the invention, including, but not limited to, such activityas would be sufficient to restore normal base-line levels and suchactivity as would be sufficient to elevate the activity above normalbase-line levels.

[0044] As mentioned above, Nitric Oxide Synthase activity is involved inmany conditions, including stroke, pulmonary hypertension, impotence,heart failure, gastric and esophageal motility disorders, kidneydisorders such as kidney hypertension and progressive renal disease,insulin deficiency, hypoxia-induced conditions, etc. In one embodimentof the invention the decrease in endothelial cell Nitric Oxide Synthaseactivity is cytokine induced. Cytokines are soluble polypeptidesproduced by a wide variety of cells that control gene activation andcell surface molecule expression. They play an essential role in thedevelopment of the immune system and thus in the development of animmune response. However, besides their numerous beneficial properties,they have also been implicated in the mechanisms for the development ofa variety of inflammatory diseases. For example, the cytokines TNF-a andIL-1 are thought to be part of the disease causing mechanism ofnon-cholesterol induced atherosclerosis, transplant arterial sclerosis,rheumatoid arthritis, lupus, scleroderma, emphysema, etc. Subjects ofsuch disorders exhibit lower levels of endothelial cell Nitric OxideSynthase activity (which is thus “cytokine induced”), and may benefitfrom therapy using the agents of the instant invention.

[0045] One important embodiment of the invention is treatment ofischemic stroke. Ischemic stroke (ischemic cerebral infarction) is anacute neurologic injury that results from a decrease in the blood flowinvolving the blood vessels of the brain. Ischemic stroke is dividedinto two broad categories, thrombotic and embolic.

[0046] A surprising finding was made in connection with the treatment ofischemic stroke. In particular, it was discovered that treatmentaccording to the invention can reduce the brain injury that follows anischemic stroke. Brain injury reduction, as demonstrated in the examplesbelow, can be measured by determining a reduction in infarct size in thetreated versus the control groups. Likewise, functional tests measuringneurological deficits provided further evidence of reduction in braininjury in the treated animals versus the controls. Cerebral blood flowalso was better in the treated animals versus the controls. Thus, in thevarious accepted models of brain injury following stroke, a positiveeffect was observed in the treated animals versus the control animals.It is believed that all of the foregoing positive results areattributable to the upregulation of endothelial cell Nitric OxideSynthase activity, which is believed demonstrated in the examples below.

[0047] An important embodiment of the invention is treatment of asubject with an abnormally elevated risk of an ischemic stroke. As usedherein, subjects having an abnormally elevated risk of an ischemicstroke are a category determined according to conventional medicalpractice. This category includes, for example, subjects which are havingelected vascular surgery. Typically, the risk factors associated withcardiac disease are the same as are associated with stroke. The primaryrisk factors include hypertension, hypercholesterolemia, and smoking. Inaddition, atrial fibrillation or recent myocardial infarction areimportant risk factors.

[0048] The treatment of stroke can be for patients who have experienceda stroke or can be a prophylactic treatment. If prophylactic, then thetreatment is for subjects having an abnormally elevated risk of anischemic stroke, as described above. If the subject has experienced astroke, then the treatment can include acute treatment. Acute treatmentmeans administration of the agents that disrupt actin cytoskeletalorganization at the onset of symptoms of the condition or at the onsetof a substantial change in the symptoms of an existing condition.

[0049] Another important embodiment of the invention is treatment ofpulmonary hypertension. Pulmonary hypertension is a diseasecharacterized by increased pulmonary arterial pressure and pulmonaryvascular resistance. Hypoxemia, hypocapnia, and an abnormal diffusingcapacity for carbon monoxide are almost invariable findings of thedisease. Additionally, according to the present invention, patients withpulmonary hypertension also have reduced levels of ecNOS expressionand/or activity in their pulmonary vessels. Traditionally, the criteriafor subjects with, or at risk for pulmonary hypertension are defined onthe basis of clinical and histological characteristics according toHeath and Edwards (Circulation, 1958, 18:533-547).

[0050] Subjects may be treated prophylactically to reduce the risk ofpulmonary hypertension or subjects with pulmonary hypertension may betreated long term and/or acutely. If the treatment is prophylactic, thenthe subjects treated are those with an abnormally elevated risk ofpulmonary hypertension. A subject with an abnormally elevated risk ofpulmonary hypertension is a subject with chronic exposure to hypoxicconditions, a subject with sustained vasoconstriction, a subject withmultiple pulmonary emboli, a subject with cardiomegaly and/or a subjectwith a family history of pulmonary hypertension.

[0051] Another important embodiment of the invention involves treatinghypoxia-induced conditions. Hypoxia as used herein is defined as thedecrease below normal levels of oxygen in a tissue. Hypoxia can resultfrom a variety of circumstances, but most frequently results fromimpaired lung function. Impaired lung function can be caused byemphysema, cigarette smoking, chronic bronchitis, asthma, infectiousagents, pneumonitis (infectious or chemical), lupus, rheumatoidarthritis, inherited disorders such as cystic fibrosis, obesity,α₁-antitrypsin deficiency and the like. It also can result from non-lungimpairments such as from living at very high altitudes. Hypoxia canresult in pulmonary vasoconstriction via inhibition of ecNOS activity.

[0052] Another important embodiment of the invention is the treatment ofheart failure. Heart failure is a clinical syndrome of diverseetiologies linked by the common denominator of impaired heart pumpingand is characterized by the failure of the heart to pump bloodcommensurate with the requirements of the metabolizing tissues, or to doso only from an elevating filling pressure.

[0053] In certain aspects of the invention, agents that disrupt actincytoskeletal organization are administered to subjects that wouldbenefit from increased endothelial cell Nitric Oxide Synthase activity.The administration of one or more agents that disrupt actin cytoskeletalorganization is in an amount(s) effective to increase endothelial cellNitric Oxide Synthase activity in tissue of the subject, provided thatthe agent that disrupts actin cytoskeletal organization used is not arho GTPase function inhibitor (See later discussion). In certainembodiments, the subject is both nonhypercholesterolemic andnonhypertriglyceridemic, i.e., nonhyperlipidemic. Such subjects arethought to benefit mostly from the treatments of the invention, but thetreatments do not necessarily exclude hyperlipidemic andhypercholesterolemic subjects.

[0054] A nonhypercholesterolemic subject is one that does not fit thecurrent criteria established for a hypercholesterolemic subject. Anonhypertriglyceridemic subject is one that does not fit the currentcriteria established for a hypertriglyceridemic subject (See, e.g.,Harrison's Principles of Experimental Medicine, 13th Edition,McGraw-Hill, Inc., N.Y.). Hypercholesterolemic subjects andhypertriglyceridemic subjects are associated with increased incidence ofpremature coronary heart disease. A hypercholesterolemic subject has anLDL level of >160 mg/dL or >130 mg/dL and at least two risk factorsselected from the group consisting of male gender, family history ofpremature coronary heart disease, cigarette smoking (more than 10 perday), hypertension, low HDL (<35 mg/dL), diabetes mellitus,hyperinsulinemia, abdominal obesity, high lipoprotein (a), and personalhistory of cerebrovascular disease or occlusive peripheral vasculardisease. A hypertriglyceridemic subject has a triglyceride (TG) levelof >250 mg/dL. Thus, a hyperlipidemic subject is defined as one whosecholesterol and triglyceride levels equal or exceed the limits set asdescribed above for both the hypercholesterolemic andhypertriglyceridemic subjects.

[0055] The invention involves treatment of the foregoing conditionsusing agents that disrupt actin cytoskeletal organization. Actincomprises a large proportion of the cytoplasmic proteins of many cells.Actin is present primarily in its globular form (G-actin), a singlepolypeptide 375 amino acids long, and is associated with one molecule ofnon-covalently bound ATP. The terminal phosphate of the ATP ishydrolysed after the actin polymerizes to form actin filaments (fibrousactin or F-actin). Actin filaments consist of a tight-helix of uniformlyoriented actin monomers. They are polar structures, with twostructurally different ends, and form the “core” of the actincytoskeleton. An actin cytoskeleton is thus a three dimensionalstructure that results from the interaction between actin filaments andother molecules that associate with the actin filaments (e.g.,cross-linking proteins such as filamin). The actin cytoskeleton mediatesa variety of biological functions in all eukaryotic cells. In additionto providing a structural framework around which cell shape and polarityare defined, its dynamic properties provide the driving force for cellsto move and to divide.

[0056] According to the present invention, it has been discovered thatagents which disrupt actin cytoskeletal organization control endothelialcell Nitric Oxide Synthase activity. In particular, agents that disruptactin cytoskeletal organization upregulate endothelial cell Nitric OxideSynthase activity.

[0057] According to the present invention, “agents that disrupt actincytoskeletal organization” are compounds, natural or synthetic, thatinterfere with actin cytoskeletal organization. Typically such agentswill interfere, for example, with stress fiber formation (contractilebundles of actin filaments and myosin), and/or focal contact (oradhesion plaque) assembly and upregulate endothelial cell Nitric OxideSynthase activity. The effects of such agents in a cell or in a tissueon actin cytoskeletal organization can be measured according to any artrecognized method. For example, a direct measure would be to performphalloidin staining (Sigma) on intact cells. A person of ordinary skillin the art could then determine (and quantitate) the effects of theagents of the invention by examining, for example, the structure of thestained actin stress-fibers and comparing such structure with the onewhich is normal and characteristic of an untreated cell.

[0058] Agents that disrupt actin cytoskeletal organization can exerttheir effects at different levels and thus comprise different categoriesof agents useful for practicing the present invention. The differentcategories include agents from those that disrupt actin cytoskeletalorganization at the nucleic acid level to agents that disrupt actincytoskeletal organization at the protein level.

[0059] Agents that disrupt actin cytoskeletal organization at thenucleotide level include chemicals, antisense nucleic acids, antibodies,catalytic nucleic acids including ribozymes, and proteins whichinterfere with the expression of a gene that encodes a polypeptide whichis a component of the actin cytoskeleton. Such exemplary polypeptidesinclude but are not limited to actin, myosin, tropomyosin, troponin,titin, nebulin, α-actinin, myomesin, C protein, filamin, talin,vinculin, capping protein, fibronectin receptor, ezrin, radixin, moiesinand the like.

[0060] Agents that disrupt actin cytoskeletal organization at theprotein level include organic molecules that inhibit or alter theformation and organization of the actin cytoskeleton by interfering(e.g., via antibody binding, etc.) or altering (e.g., viapost-translational modification) an individual component of the actincytoskeleton. Specifically included are proteins, peptides and lipidderivatives. Antibodies include polyclonal and monoclonal antibodies,prepared according to conventional methodology.

[0061] Significantly, as is well-known in the art, only a small portionof an antibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

[0062] Within the antigen-binding portion of an antibody, as iswell-known in the art, there are complementarity determining regions(CDRs), which directly interact with the epitope of the antigen, andframework regions (FRs), which maintain the tertiary structure of theparatope (see, in general, Clark, 1986; Roitt, 1991). In both the heavychain Fd fragment and the light chain of IgG immunoglobulins, there arefour framework regions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

[0063] It is now well-established in the art that the non-CDR regions ofa mammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

[0064] Thus, as will be apparent to one of ordinary skill in the art,the present invention also provides for F(ab′)₂, Fab, Fv and Fdfragments; chimeric antibodies in which the Fc and/or FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences; chimeric F(ab′)₂ fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or light chainCDR3 regions have been replaced by homologous human or non-humansequences; chimeric Fab fragment antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences; and chimeric Fd fragmentantibodies in which the FR and/or CDR1 and/or CDR2 regions have beenreplaced by homologous human or non-human sequences. The presentinvention also includes so-called single chain antibodies.

[0065] In certain embodiments, agents that disrupt actin cytoskeletalorganization include myosin light chain kinase (MLCK-Ser/Thr kinases)inhibitors, myosin light chain phosphatase (MLCP) stimulators, proteinkinase N (PKN) inhibitors, phospatidylinositol 4-phosphate 5-kinase(PIP5K) inhibitors, and cytochalasin D. Exemplary myosin light chainkinase inhibitors include BDM [2,3-butanedione 2-monoxime], ML-7[1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1 ,4-diazepinehydrochloride], ML-9[1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepinehydrochloride], wortmannin, H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride], Fasudil (HA1077)[Hexahydro-1-(5-isoquinolinesulphonyl)-1H-1,4- diazepine], W7[N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide] and A-3[N-(6-Aminoethyl)-5-chloro-1-naphthalenesulfonamide]. In preferredembodiments, agents that disrupt actin cytoskeletal organization includeBDM, ML-7, H-7 and cytochalasin D. Exemplary PKN inhibitors include“dominant negative” PKN peptides and purine analogues such as6-thioguanine. Exemplary PIP5K inhibitors include “dominant negative”PIP5K peptides. Exemplary MLCP stimulators include nucleic acids thatencode functional MLCP proteins and peptides (i.e., maintain thephosphatase activity of MLCP) and that are overexpressed (via anexpression vector) in the cells of interest of a subject according tothe invention.using genetic approaches well known in the art.Cytochalasin D is a preferred agent of the invention that belongs to thefamily of mold metabolites called cytochalasins. Cytochalasin D isthought to exert its function as an agent that disrupts actincytoskeletal organization by affecting actin polymerization. Othermembers of the cytochalasin family share this property (e.g.,Cytochalasin B), and are thus useful according to the invention.

[0066] Examples of agents that disrupt actin cytoskeletal organizationalso include “dominant negative” polypeptides of the polypeptidecomponents of the actin cytoskeleton, some of which are exemplifiedabove. A dominant negative polypeptide is an inactive variant of aprotein, which, by interacting with the cellular machinery, displaces anactive protein from its interaction with the cellular machinery orcompetes with the active protein, thereby reducing the effect of theactive protein. For example, a dominant negative receptor which binds aligand but does not transmit a signal in response to binding of theligand can reduce the biological effect of expression of the ligand.Likewise, a dominant negative catalytically-inactive kinase whichinteracts normally with target proteins but does not phosphorylate thetarget proteins can reduce phosphorylation of the target proteins inresponse to a cellular signal. Similarly, a dominant negativetranscription factor which binds to a promoter site in the controlregion of a gene but does not increase gene transcription can reduce theeffect of a normal transcription factor by occupying promoter bindingsites without increasing transcription.

[0067] The end result of the application of or expression of a dominantnegative polypeptide is a reduction in function of active proteins. Oneof ordinary skill in the art can assess the potential for a dominantnegative variant of a protein, and using standard mutagenesis techniquesto create one or more dominant negative variant polypeptides. Forexample, given the teachings contained herein and in the art, one ofordinary skill in the art can modify the sequence of a polypeptide (orthe gene encoding a polypeptide) of an actin cytoskeletal component (asdescribed earlier, e.g., actin, myosin, filamin, etc.) by site-specificmutagenesis, scanning mutagenesis, partial gene deletion or truncation,and the like. See, e.g., U.S. Pat. No. 5,580,723 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, 1989. The skilled artisan then can test thepopulation of mutagenized polypeptides for diminution in a selectedactivity (e.g., impaired myosin light chain phosphorylation andupregulation of ecNOS activity) and/or for retention of such anactivity. Other similar methods for creating and testing dominantnegative variants of a protein will be apparent to one of ordinary skillin the art.

[0068] Other examples of agents that disrupt actin cytoskeletalorganization include polypeptides which bind to components of the actincytoskeleton and to complexes of the components of the actincytoskeleton and binding partners. The invention, therefore, embracespeptide binding agents which, for example, can be antibodies orfragments of antibodies having the ability to selectively bind tocomponents of the actin cytoskeleton. Antibodies include polyclonal andmonoclonal antibodies, prepared according to conventional methodology.

[0069] A rho GTPase is a small, membrane-bound, Ras-related GTP-bindingprotein that functions by binding and hydrolyzing GTP. Rho GTPasesfunction as molecular switches, cycling between an inactive GDP-boundconformation and an active GTP-bound conformation. According to thepresent invention, “rho GTPase function inhibitors” are compounds,natural or synthetic, that inhibit the normal function and localizationof rho GTPases (i.e., impair GTP binding by rho GTPases) and upregulateendothelial cell Nitric Oxide Synthase activity. Such compounds caninhibit rho GTPase function at different levels and thus comprisedifferent categories of agents useful for practicing the presentinvention. The different categories include agents from those thatinhibit rho GTPases at the nucleic acid level to agents that inhibit rhoGTPases at the protein level.

[0070] Agents that inhibit rho GTPases at the nucleotide level includechemicals, antisense nucleic acids, antibodies, catalytic nucleic acidsincluding ribozymes, and proteins which repress expression of a rhoGTPase gene locus.

[0071] Agents that inhibit rho GTPases at the protein level includeorganic molecules that alter the intrinsic GTPase activity of the rhoGTP-binding protein, organic molecules that inhibit GDP/GTP exchange,and organic molecules that inhibit or alter post-translationalmodifications of rho GTPases. Specifically included are proteins,peptides and lipid derivatives.

[0072] Examples of agents that inhibit or reduce the intrinsic GTPaseactivity of a rho GTP-binding protein include cyclosporin, and “dominantnegative” polypeptides of the rho GTPase. A dominant negativepolypeptide is as described previously.

[0073] Dominant negative rho GTPase proteins include variants in which aportion of the GTP catalytic site has been mutated or deleted to reduceor eliminate GTP binding. Other examples include rho GTPase variants inwhich the conserved CAAX motif at their carboxy-terminus has beenmutated or deleted to reduce or eliminate post-tranlationalmodification. (C, cysteine; A, aliphatic amino acid; X, any amino acid).One of ordinary skill in the art can readily prepare such modifications.Examples of dominant negative rho GTPase peptides are described in theExamples section and include N19RhoA and CAAXRhoA.

[0074] Other examples of agents that inhibit or reduce the intrinsicGTPase activity of a rho GTP-binding protein include polypeptides whichbind to rho GTPase polypeptides and to complexes of rho GTPasepolypeptides and binding partners. The invention, therefore, embracespeptide binding agents which, for example, can be antibodies orfragments of antibodies having the ability to selectively bind to rhoGTPase polypeptides. Antibodies include polyclonal and monoclonalantibodies, prepared according to conventional methodology.

[0075] Examples of agents that inhibit the GDP/GTP exchange includeproteins and peptides that inhibit GDP-dissociation such as Ly-GDI andRhoGDI-3. Preferably, using genetic approaches well known in the art,such proteins and peptides can be overexpressed (via an expressionvector) in the cells of interest of a subject according to theinvention.

[0076] Post-translational modifications of rho GTPases are important inthat they are necessary for the proper attachment (and thus function) ofthe rho GTPases to the cell membrane. If rho GTPase polypeptides cannotbe properly modified (or if they are overmodified), they accumulate inthe cytosol and are rendered inactive. Examples of agents that inhibitpost-translational modifications of rho GTPases includegeranylgeranylation inhibitors and guanine nucleotide exchangeinhibitors.

[0077] Geranylgeranylation inhibitors are compounds (natural orsynthetic) that interfere with the geranylgeranylation of rho GTPases,and include proteins, peptides and lipid derivatives. Thus,geranylgeranylation inhibition of rho GTPases can occur either bypreventing geranylgeranyl-pyrophosphate synthesis, or by inhibiting theenzyme geranylgeranyl transferase (GGT) which attachesgeranylgeranyl-pyrophosphate to the CAAX motif of rho GTPases.Geranylgeranyl-pyrophosphate synthesis inhibition can be performed bypreventing or inhibiting the formation of any of the intermediates inthe geranylgeranyl-pyrophosphate synthesis pathway. Examples includemevalonate inhibitors, isopentenyl-pyrophosphate inhibitors,geranyl-pyrophosphate inhibitors, farnesyl-pyrophosphate inhibitors andgeranylgeranyl-pyrophosphate inhibitors. Examples of such compoundsinclude farnesyl-transferase inhibitors disclosed in U.S. Pat. Nos.5,705,686 and 5,602,098, inhibitors of geranylgeranyl-transferasedisclosed in U.S. Pat. No. 5,470,832, the disclosure of which isincorporated herein by reference, and a-hydroxyfarnesylphosphonic acid.Additional geranylgeranyl-transferase inhibitors include GGTI-298(Finder, J D et al., J Biol Chem, 1997, 272:13484-13488).

[0078] Guanine nucleotide exchange inhibitors are agents that alsopost-translationaly modify and inactivate rho GTPases. They includebacterial protein toxins that ADP-ribosylate or glucosylate rho GTPases,or compounds that inhibit rho GTPase-specific guanine nucleotideexchange factor (GEF). Preferred such agents according to the inventioninclude Clostridium botulinum C3 transferase. The C3 transferaseenzymatically catalyses the transfer of ADP from NADH to Asp-41 of rho,rendering the rho GTPase resistant to GTP/GDP exchange by the rhoGTPase-specific guanine nucleotide exchange factors (GEFs). (See theExamples section also). The C3 transferase is administered in proteinform, or more preferably, its cDNA is expressed using an expressionvector in the cells of interest of a subject according to the invention.Rho GTPase-specific guanine nucleotide exchange factor inhibitorsinclude chemicals, antisense nucleic acids, antibodies, catalyticnucleic acids including ribozymes, proteins which repress expression ofa rho GTPase-specific guanine nucleotide exchange factor gene locus,proteins, peptides (including dominant-negative peptides andantibodies), and the like.

[0079] According to the invention, agents that disrupt actincytoskeletal organization are used excluding rho GTPase functioninhibitors as agents useful in upregulating ecNOS activity. Theinvention can involve use of a rho GTPase function inhibitor (includinga HMG-CoA reductase inhibitor), however, only if used together with anagent that disrupts actin cytoskeletal organization other than a rhoGTPase function inhibitor.

[0080] HMG-CoA reductase inhibitors inhibit post-translationalmodifications of rho GTPases by preventing mevalonate synthesis andconsequently geranylgeranylpyrophosphate synthesis, an isoprenoid thatis attached to the CAAX motif of rho GTPases. Examples of HMG-CoAreductase inhibitors include some which are commercially available, suchas simvastatin (U.S. Pat. No. 4, 444,784), lovastatin (U.S. Pat. No.4,231,938), pravastatin sodium (U.S. Pat. No. 4,346,227), fluvastatin(U.S. Pat. No. 4,739,073), atorvastatin (U.S. Pat. No. 5,273,995),cerivastatin, and numerous others described in U.S. Pat. No. 5,622,985,U.S. Pat. No. 5,135,935, U.S. Pat. No. 5,356,896, U.S. Pat. No.4,920,109, U.S. Pat. No. 5,286,895, U.S. Pat. No. 5,262,435, U.S. Pat.No. 5,260,332, U.S. Pat. No. 5,317,031, U.S. Pat. No. 5,283,256, U.S.Pat. No. 5,256,689, U.S. Pat. No. 5,182,298, U.S. Pat. No. 5,369,125,U.S. Pat. No. 5,302,604, U.S. Pat. No. 5,166,171, U.S. Pat. No.5,202,327, U.S. Pat. No. 5,276,021, U.S. Pat. No. 5,196,440, U.S. Pat.No. 5,091,386, U.S. Pat. No. 5,091,378, U.S. Pat. No. 4,904,646, U.S.Pat. No. 5,385,932, U.S. Pat. No. 5,250,435, U.S. Pat. No. 5,132,312,U.S. Pat. No. 5,130,306, U.S. Pat. No. 5,116,870, U.S. Pat. No.5,112,857, U.S. Pat. No. 5,102,911, U.S. Pat. No. 5,098,931, U.S. Pat.No. 5,081,136, U.S. Pat. No. 5,025,000, U.S. Pat. No. 5,021,453, U.S.Pat. No. 5,017,716, U.S. Pat. No. 5,001,144, U.S. Pat. No. 5,001,128,U.S. Pat. No. 4,997,837, U.S. Pat. No. 4,996,234, U.S. Pat. No.4,994,494, U.S. Pat. No. 4,992,429, U.S. Pat. No. 4,970,231, U.S. Pat.No. 4,968,693, U.S. Pat. No. 4,963,538, U.S. Pat. No. 4,957,940, U.S.Pat. No. 4,950,675, U.S. Pat. No. 4,946,864, U.S. Pat. No. 4,946,860,U.S. Pat. No. 4,940,800, U.S. Pat. No. 4,940,727, U.S. Pat. No.4,939,143, U.S. Pat. No. 4,929,620, U.S. Pat. No. 4,923,861, U.S. Pat.No. 4,906,657, U.S. Pat. No. 4,906,624 and U.S. Pat. No. 4,897,402, thedisclosures of which patents are incorporated herein by reference.

[0081] Other rho GTPase function inhibitors not described in the abovecategories and useful according to the invention include agents thatinhibit rho GTPase activation via a receptor-mediated signaling pathway.Such agents include protein kinase C inhibitors, Gq protein inhibitors(e.g., C-terminal antibodies, dominant-negative Gq mutants, etc.),tyrosine kinase inhibitors (e.g., genistein, etc.), tyrosine phosphatasestimulators, GTPase-activating protein stimulators, inhibitors ofintegrins and adhesion molecules, adapter protein (Shc and Sos)inhibitors, and Pleckstrin homology domains which bind G-protein bg.

[0082] The invention also involves the co-administration of agents thatare not agents that disrupt actin cytoskeletal organization but that canact cooperatively, additively or synergistically with such agents thatdisrupt actin cytoskeletal organization to increase ecNOS activity.Thus, ecNOS substrates which are converted by ecNOS to nitric oxide canbe co-administered with the agents that disrupt actin cytoskeletalorganization according to the invention. Such ecNOS substrates may benatural or synthetic, although the preferred substrate is L-arginine.

[0083] Likewise, there are other agents besides agents that disruptactin cytoskeletal organization, that are not substrates of ecNOS, andthat can increase ecNOS activity. Examples of categories of such agentsare estrogens and ACE inhibitors. Estrogens are a well defined categoryof molecules known by those of ordinary skill in the art, and will notbe elaborated upon further herein. All share a high degree of structuralsimilarity. ACE inhibitors also have been well characterized, althoughthey do not always share structural homology.

[0084] Angiotensin converting enzyme, or ACE, is an enzyme whichcatalyzes the conversion of angiotensin I to angiotensin II. ACEinhibitors include amino acids and derivatives thereof, peptides,including di and tri peptides and antibodies to ACE which intervene inthe renin-angiotensin system by inhibiting the activity of ACE therebyreducing or eliminating the formation of pressor substance angiotensinII. ACE inhibitors have been used medically to treat hypertension,congestive heart failure, myocardial infarction and renal disease.Classes of compounds known to be useful as ACE inhibitors includeacylmercapto and mercaptoalkanoyl prolines such as captopril (U.S. Pat.No. 4,105,776) and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyldipeptides such as enalapril (U.S. Pat. No. 4,374,829), lisinopril (U.S.Pat. No. 4,374,829), quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S.Pat. No. 4,587,258), and perindopril (U.S. Pat. No. 4,508,729),carboxyalkyl dipeptide mimics such as cilazapril (U.S. Pat. No.4,512,924) and benazapril (U.S. Pat. No. 4,410,520), phosphinylalkanoylprolines such as fosinopril (U.S. Pat. No. 4,337,201) and trandolopril.

[0085] Estrogens upregulate Nitric Oxide Synthase expression whereas ACEinhibitors do not affect expression, but instead influence theefficiency of the action of Nitric Oxide Synthase on L-arginine. Thus,activity can be increased in a variety of ways. In general, activity isincreased by the reductase inhibitors of the invention by increasing theamount of the active enzyme present in a cell versus the amount presentin a cell absent treatment with the reductase inhibitors according tothe invention.

[0086] The invention also involves the co-administration of “at leastone different agent that disrupts actin cytoskeletal organization”(second agent that disrupts actin cytoskeletal organization) that canact cooperatively, additively or synergistically with a first agent thatdisrupts actin cytoskeletal organization of the invention to increaseecNOS activity. Thus, “at least one different agent that disrupts actincytoskeletal organization” is meant to include one or more agent(s) thatdisrupts actin cytoskeletal organization that is (are) different to thefirst agent that disrupts actin cytoskeletal organization of theinvention and can include a HMG-CoA reductase inhibitor and/or a rhoGTPase function inhibitor. In one embodiment, when the agent thatdisrupts actin cytoskeletal organization according to the invention isco-administered in combination with “at least one different agent thatdisrupts actin cytoskeletal organization” and the “at least onedifferent agent that disrupts actin cytoskeletal organization” is aHMG-CoA reductase inhibitor, the subject is nonhypercholesterolemic.

[0087] The agents that disrupt actin cytoskeletal organization areadministered in effective amounts. In general, an effective amount isany amount that can cause an increase in Nitric Oxide Synthase activityin a desired cell or tissue, and preferably in an amount sufficient tocause a favorable phenotypic change in a condition such as a lessening,alleviation or elimination of a symptom or of a condition.

[0088] In general, an effective amount is that amount of apharmaceutical preparation that alone, or together with further doses orco-administration of other agents, produces the desired response. Thismay involve only slowing the progression of the disease temporarily,although more preferably, it involves halting the progression of thedisease permanently or delaying the onset of or preventing the diseaseor condition from occurring. This can be monitored by routine methods.Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 50-500mg/kg will be suitable, preferably orally and in one or severaladministrations per day.

[0089] Such amounts will depend, of course, on the particular conditionbeing treated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. Lower doses willresult from certain forms of administration, such as intravenousadministration. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple dosesper day are contemplated to achieve appropriate systemic levels ofcompounds. It is preferred generally that a maximum dose be used, thatis, the highest safe dose according to sound medical judgment. It willbe understood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

[0090] The agents that disrupt actin cytoskeletal organization usefulaccording to the invention may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

[0091] The pharmaceutical compositions may contain suitable bufferingagents, including: acetic acid in a salt; citric acid in a salt; boricacid in a salt; and phosphoric acid in a salt.

[0092] The pharmaceutical compositions also may contain, optionally,suitable preservatives, such as: benzalkonium chloride; chlorobutanol;parabens and thimerosal.

[0093] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular drug selected,the severity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis.

[0094] The pharmaceutical compositions may conveniently be presented inunit dosage form and may be prepared by any of the methods well-known inthe art of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

[0095] Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

[0096] Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of reductase inhibitors, which ispreferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono-ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

[0097] Other delivery systems can include time-release, delayed releaseor sustained release delivery systems. Such systems can avoid repeatedadministrations of the active compound, increasing convenience to thesubject and the physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer base systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di-and tri-glycerides; hydrogelrelease systems; sylastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the active compound iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) difusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

[0098] Use of a long-term sustained release implant may be desirable.Long-term release, are used herein, means that the implant isconstructed and arranged to delivery therapeutic levels of the activeingredient for at least 30 days, and preferably 60 days. Long-termsustained release implants are well-known to those of ordinary skill inthe art and include some of the release systems described above.

[0099] According to another aspect of the invention, a method forincreasing blood flow in a tissue of a subject is provided. The methodinvolves administering to a subject in need of such treatment a firstagent that disrupts actin cytoskeletal organization in an amounteffective to increase endothelial cell Nitric Oxide Synthase activity inthe tissue of the subject, provided that the first agent is not a rhoGTPase function inhibitor or fasudil. Fasudil (a substitutedisoquinolinesulfonyl compound- also known as HA1077), described in U.S.Pat. No. 4,678,783 as a compound with vasodilating properties, was notknown to act as an agent that disrupts actin cytoskeletal organizationresulting in increased ecNOS expression prior to the present invention.

[0100] In important embodiments a second agent is co-administered to asubject with a condition treatable by the second agent in an amounteffective to treat the condition, whereby the delivery of the secondagent to a tissue of the subject is enhanced as a result of theincreased blood flow from administering the first agent of the invention(an agent that disrupts actin cytoskeletal organization). The “secondagent” may be any pharmacological compound or diagnostic agent, asdesired.

[0101] Examples of catagories of pharmaceutical agents include:adrenergic agent; adrenocortical steroid; adrenocortical suppressant;alcohol deterrent; aldosterone antagonist; amino acid; ammoniadetoxicant; anabolic; analeptic; analgesic; androgen; anesthesia,adjunct to; anesthetic; anorectic; antagonist; anterior pituitarysuppressant; anthelmintic; anti-acne agent; anti-adrenergic;anti-allergic; anti-amebic; anti-androgen; anti-anemic; anti-anginal;anti-anxiety; anti-arthritic; anti-asthmatic; anti-atherosclerotic;antibacterial; anticholelithic; anticholelithogenic; anticholinergic;anticoagulant; anticoccidal; anticonvulsant; antidepressant;antidiabetic; antidiarrheal; antidiuretic; antidote; anti-emetic;anti-epileptic; anti-estrogen; antifibrinolytic; antifungal;antiglaucoma agent; antihemophilic; antihemorrhagic; antihistamine;antihyperlipidemia; antihyperlipoproteinemic; antihypertensive;anti-infective; anti-infective, topical; anti-inflammatory;antikeratinizing agent; antimalarial; antimicrobial; antimigraine;antimitotic; antimycotic, antinauseant, antineoplastic, antineutropenic,antiobessional agent; antiparasitic; antiparkinsonian; antiperistaltic,antipneumocystic; antiproliferative; antiprostatic hypertrophy;antiprotozoal; antipruritic; antipsychotic; antirheumatic;antischistosomal; antiseborrheic; antisecretory; antispasmodic;antithrombotic; antitussive; anti-ulcerative; anti-urolithic; antiviral;appetite suppressant; benign prostatic hyperplasia therapy agent; bloodglucose regulator; bone resorption inhibitor; bronchodilator; carbonicanhydrase inhibitor; cardiac depressant; cardioprotectant; cardiotonic;cardiovascular agent; choleretic; cholinergic; cholinergic agonist;cholinesterase deactivator; coccidiostat; cognition adjuvant; cognitionenhancer; depressant; diagnostic aid; diuretic; dopaminergic agent;ectoparasiticide; emetic; enzyme inhibitor; estrogen; fibrinolytic;fluorescent agent; free oxygen radical scavenger; gastrointestinalmotility effector; glucocorticoid; gonad-stimulating principle; hairgrowth stimulant; hemostatic; histamine H2 receptor antagonists;hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive;imaging agent; immunizing agent; immunomodulator; immunoregulator;immunostimulant; immunosuppressant; impotence therapy adjunct;inhibitor; keratolytic; LNRH agonist; liver disorder treatment;luteolysin; memory adjuvant; mental performance enhancer; moodregulator; mucolytic; mucosal protective agent; mydriatic; nasaldecongestant; neuromuscular blocking agent; neuroprotective; NMDAantagonist; non-hormonal sterol derivative; oxytocic; plasminogenactivator; platelet activating factor antagonist; platelet aggregationinhibitor; post-stroke and post-head trauma treatment; potentiator;progestin; prostaglandin; prostate growth inhibitor; prothyrotropin;psychotropic; pulmonary surface; radioactive agent; regulator; relaxant;repartitioning agent; scabicide; sclerosing agent; sedative;sedative-hypnotic; selective adenosine Al antagonist; serotoninantagonist; serotonin inhibitor; serotonin receptor antagonist; steroid;stimulant; suppressant; symptomatic multiple sclerosis; synergist;thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer;treatment of amyotrophic lateral sclerosis; treatment of cerebralischemia; treatment of Paget's disease; treatment of unstable angina;uricosuric; vasoconstrictor; vasodilator; vulnerary; wound healingagent; xanthine oxidase inhibitor.

[0102] In another aspect of the invention, the agent that disrupts actincytoskeletal organization is “co-administered,” which means administeredsubstantially simultaneously with another agent. By substantiallysimultaneously, it is meant that the agent that disrupts actincytoskeletal organization is administered to the subject close enough intime with the administration of the other agent, whereby the twocompounds may exert an additive or even synergistic effect, i.e. onincreasing ecNOS activity or on delivering a second agent to a tissuevia increased blood flow.

EXAMPLES

[0103] “Upregulation of endothelial cell Nitric Oxide Synthase by HMGCoA Reductase Inhibitors”

[0104] Experimental Procedures

[0105] All standard culture reagents were obtained from JRH Bioscience(Lenexa, Kans.). Unless indicated otherwise, all reagents were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). [a-³²P]CTP (3000 Ci/mmol) wassupplied by New England Nuclear. Purified human LDL was obtained fromCalbiochem (San Diego, Calif.; lot#730793) and Biomedical TechnologiesInc. (Stoughton, Mass.; lot#9030197). The level of endotoxin wasdetermined by the chromogenic Limulus amebocyte assay (BioWhittakerInc., Walkersville, Md.). The antibody detection kit (EnhancedChemiluminescence) and the nylon nucleic acid (Hybond) and protein(PVDF) transfer membranes were purchased from Amersham Corp. (ArlingtonHeights, Ill.). Simvastatin and lovastatin were obtained from Merck,Sharp, and Dohme, Inc. (West Point, Pa.). Since endothelial cells lacklactonases to process simvastatin and lovastatin to their active forms,these HMG-CoA reductase inhibitors were chemically activated prior totheir use with methods well known in the art and as previously described(Laufs, U et al., J Biol Chem, 1997, 272:31725-31729).

[0106] Cell Culture:

[0107] Human endothelial cells were harvested from saphenous veins andcultured as described (15). For transfection studies, bovine aorticendothelial cells of less than 3 passages were cultured in a growthmedium containing DMEM (Dulbecco's Modified Eagle's Medium), 5 mmol/LL-glutamine (Gibco), and 10% fetal calf serum (Hyclone Lot#1114577). Forall experiments, the endothelial cells were placed in 10%lipoprotein-deficient serum (Sigma, Lot#26H9403 1) for 48 h prior totreatment conditions. In the indicated experiments, endothelial cellswere pretreated with actinomycin D (5 mg/ml) for 1 h prior to treatmentwith ox-LDL and/or simvastatin. Cellular viability as determined by cellcount, morphology, and Trypan blue exclusion was maintained for alltreatment conditions.

[0108] Preparation of LDL:

[0109] The LDL was prepared by discontinuous ultracentrifugationaccording to the method of Chung et al. with some modification (MethodsEnzymol, 1984, 128:181-209). Fresh plasma from a single donor wasanticoagulated with heparin and filtered through a Sephadex G-25 columnequilibrated with PBS. The density was adjusted to 1.21 g/ml by additionof KBr (0.3265 g/ml plasma). A discontinuous NaCl/KBr gradient wasestablished in Beckman Quick-Seal centrifuge tubes (5.0 ml capacity) bylayering 1.5 ml of density-adjusted plasma under 3.5 ml of 0.154 M NaClin Chelex-100-treated water (BioRad, Hercules, Calif.). Afterultracentrifugation at 443,000× g and 7° C. for 45 min in a Beckman NearVertical Tube 90 rotor (Beckman L8-80M ultracentrifuge), the yellow bandin the upper middle of the tube corresponding to LDL was removed bypuncturing with a needle and withdrawing into a syringe. The KBr wasremoved from the LDL by dialyzing with three changes of sterile PBS, pH7.4, containing 100 μg/ml polymyxin B.

[0110] The purity of the LDL samples was confirmed by SDS/polyacrylamideand cellulose acetate gel electrophoresis. Cholesterol and triglyceridecontent were determined as previously described (Liao, J K et al., JBiol Chem, 1995, 270:319-324.). The LDL protein concentration wasdetermined by the method of Lowry et al., (J Biol Chem, 1951,193:265-275.). For comparison, commercially-available LDL (BiomedicalTechnologies Inc., Stoughton, M A; Calbiochem, San Diego, Calif.) werecharacterized and used in selected experiments.

[0111] Oxidation of LDL:

[0112] Oxidized LDL was prepared by exposing freshly-isolated LDL toCuSO₄ (5-10 mM) at 37° C. for various duration (6-24 h). The reactionwas stopped by dialyzing with three changes of sterile buffer (150μmol/L NaCl, 0.01% EDTA and 100 μg/ml polymyxin B, pH 7.4) at 4° C. Thedegree of LDL oxidation was estimated by measuring the amounts ofthiobarbituric acid reactive substances (TBARS) produced using afluorescent assay for malondialdehyde as previously described (Yagi, KA, Biochem Med, 1976, 15:212-21.). The extent of LDL modification wasexpressed as nanomoles of malondialdehyde per mg of LDL protein. Onlymild to moderate ox-LDL with TBARS values between 12 and 16 nmol/mg LDLprotein (i.e. 3 to 4 nmol/mg LDL cholesterol) were used in this study.All oxidatively-modified LDL samples were used within 24 h ofpreparation.

[0113] Northern Blotting:

[0114] Equal amounts of total RNA (10-20 mg) were separated by 1.2%formaldehyde-agarose gel electrophoresis and transferred overnight ontoHybond nylon membranes. Radiolabeling of human full-length ecNOS cDNA(Verbeuren, T J et al., Circ Res, 1986, 58:552-564, Liao, J K et al., JClin Invest, 1995, 96:2661-2666) was performed using random hexamerpriming, [a-³²P]CTP, and Klenow (Pharmacia). The membranes werehybridized with the probes overnight at 45° C. in a solution containing50% formamide, 5× SSC, 2.5× Denhardt's Solution, 25 mM sodium phosphatebuffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA. All Northernblots were subjected to stringent washing conditions (0.2× SSC/0.1% SDSat 65° C.) prior to autoradiography. RNA loading was determined byrehybridization with human GAPDH probe.

[0115] Western Blotting:

[0116] Cellular proteins were prepared and separated on SDS/PAGE asdescribed (Liao, J K et al., J Biol Chem, 1995, 270:319-324).Immunoblotting was performed using a murine monoclonal antibody to humanecNOS (1:400 dilution, Transduction Laboratories, Lexington, Ky.).Immunodetection was accomplished using a sheep anti-mouse secondaryantibody (1:4000 dilution) and the enhanced chemiluminescence (ECL) kit(Amersham Corp., Arlington Heights, Ill.). Autoradiography was performedat 23° C. and the appropriate exposures were quantitated bydensitometry.

[0117] Assay for ecNOS Activity:

[0118] The ecNOS activity was determined by a modified nitrite assay aspreviously described (Misko, T P et al., Analytical Biochemistry, 1993,214:11-16, Liao, J K et al., J Clin Invest, 1995, 96:2661-2666).Briefly, endothelial cells were treated for 24 h with ox-LDL in thepresence and absence of simvastatin (0.1 to 1 mM). After treatment, themedium was removed, and the cells were washed and incubated for 24 h inphenol red-free medium. After 24 h, 300 μl of conditioned medium wasmixed with 30 μl of freshly prepared 2,3-diaminonaphthalene (1.5 mmol/LDAN in 1 mol/L HCl). The mixture was protected from light and incubatedat 20° C. for 10 min. The reaction was terminated with 15 μl of 2.8mol/L NaOH. Fluorescence of 1-(H)-naphthotriazole was measured withexcitation and emission wavelengths of 365 and 450 nm, respectively.Standard curves were constructed with known amounts of sodium nitrite.Nonspecific fluorescence was determined in the presence of LNMA (5mmol/L).

[0119] Nuclear Run-on Assay:

[0120] Confluent endothelial cells (˜5×10⁷ cells) grown in LPDS weretreated with simvastatin (1 mM) or 95%O₂ for 24 h. Nuclei were isolatedand in vitro transcription was performed as previously described (Liao,J K et al., J Clin Invest, 1995, 96:2661-2666). Equal amounts (1 mg) ofpurified, denatured full-length human ecNOS, human b-tubulin (ATCC#37855), and linearized pGEM-3z cDNA were vacuum-transferred ontonitrocellulose membranes using a slot blot apparatus (Schleicher &Schuell). Hybridization of radiolabeled mRNA transcripts to thenitrocellulose membranes was carried out at 45° C. for 48 h in a buffercontaining 50% formamide, 5× SSC, 2.5× Denhardt's solution, 25 mM sodiumphosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA. Themembranes were then washed with 1× SSC/0.1% SDS for 1 h at 65° C. priorto autoradiography for 72 h at −80° C.

[0121] Transfection Assays:

[0122] For transient transfections, bovine rather than human endothelialcells were used because of their higher transfection efficiency by thecalcium-phosphate precipitation method (12% vs<4%) (Graham, F L and Vander Erb, A J, Virology, 1973, 52:456-457). We used the human ecNOSpromoter construct, F1.LUC, which contains a −1.6 kb 5′-upstreamsequence linked to the luciferase reporter gene as described by Zhang etal. (J Biol Chem, 1995, 270:15320-15326). Bovine endothelial cells(60%-70% confluent) were transfected with 30 mg of the indicatedconstructs: p.LUC (no promoter), pSV2.LUC (SV40 early promoter), orF1.LUC. As an internal control for transfection efficiency, pCMV.bGalplasmid (10 mg) was co-transfected in all experiments. Preliminaryresults using b-galactosidase staining indicate that cellulartransfection efficiency was approximately 10% to 14%.

[0123] Endothelial cells were placed in lipoprotein-deficient serum for48 h after transfection and treated with ox-LDL (50 mg/ml, TBARS 12.4nmol/mg) in the presence and absence of simvastatin (1 mM) for anadditional 24 h. The luciferase and b-galactosidase activities weredetermined by a chemiluminescence assay (Dual-Light, Tropix, Bedford,Mass.) using a Berthold L9501 luminometer. The relative promoteractivity was calculated as the ratio of luciferase- to b-galactosidaseactivity. Each experiment was performed three times in triplicate.

[0124] Data Analysis:

[0125] Band intensities were analyzed densitometrically by the NationalInstitutes of Health Image program (Rasband, W, NIH Image program, v1.49, National Institutes of Health, Bethesda, 1993). All values areexpressed as mean i SEM compared to controls and among separateexperiments. Paired and unpaired Student's t tests were employed todetermine any significant changes in densitometric values, nitriteproduction, and promoter activities. A significant difference was takenfor P values less than 0.05.

EXAMPLE 1 Cell Culture

[0126] Relatively pure (>95%) human endothelial cell cultures wereconfirmed by their morphological features (i.e. cuboidal, cobble-stone,contact inhibited) using phase-contrast microscopy and byimmunofluorescent staining with anti-Factor VIII antibodies (Gerson, R Jet al., Am J Med, 1989, 87:28-38). For all experimental conditions,there were no observable adverse effects of ox-LDL or HMG-CoA reductaseinhibitors on cellular morphology, cell number, immunofluorescentstaining, and Trypan blue exclusion (>95%). Higher concentrations ofox-LDL (>100 mg/ml) with greater oxidative modification (i.e. TBARSvalues of >30 nmol/mg) caused vacuolization and some detachment ofendothelial cells after 24 h. Neither simvastatin (0.01 to 0.1 mmol/L)nor lovastatin (10 mmol/L) produced any noticeable adverse effects onhuman endothelial cell for up to 96 h. However, higher concentrations ofsimvastatin (>15 mmol/L) or lovastatin (>50 mmol/L) caused cytotoxicityafter 36 h, and therefore, were not used.

EXAMPLE 2 Characterization of LDL

[0127] SDS/polyacrylamide gel electrophoresis of native or unmodifiedLDL revealed a single band (˜510 kD) corresponding to ApoB-100 (data notshown). Similarly, cellulose acetate electrophoresis revealed only oneband corresponding to the presence of a single class of low-densitylipids (density of 1.02 to 1.06 g/ml). The LDL had a protein,cholesterol, and triglyceride concentration of 6.3±0.2, 2.5±0.1, and0.5±0.1 mg/ml, respectively. In contrast, lipoprotein-deficient serumwas devoid of both apoB-100 protein and low-density lipid bands, and hadnon-detectable levels of cholesterol. There was no detectable level ofendotoxin (<0.10 EU/ml) in the lipoprotein-deficient serum or ox-LDLsamples by the chromogenic Limulus amebocyte assay.

[0128] In addition, there was no apparent difference between our ownpreparation and commercially-obtained LDL samples in terms ofelectrophoretic mobility. Native LDL had a TBARS value of 0.3±0.2nmol/mg, but after exposure to human saphenous vein endothelial cells inlipoprotein-deficient media for 72 h, this value increased to 3.1±0.4nmol/mg. Copper-oxidized LDL had TBARS values ranging from 4.6±0.5 to33.1±5.2 nmol/mg. The degree of ox-LDL used in this study was mild tomoderate with TBARS value ranging from 12 to 16 nmol/mg LDL protein(i.e. 3 to 4 nmol/mg LDL cholesterol).

EXAMPLE 3 Effect of ox-LDL and HMG-CoA Reductase Inhibitors on ecNOSProtein

[0129] We have previously shown that ox-LDL (50 mg/ml) downregulatesecNOS expression (Liao, J K et al., J Biol Chem, 1995, 270:319-324).Compared to untreated cells, treatment with ox-LDL (50 mg/ml, TBARS 12.2nmol/mg) caused a 54%±6% decrease in ecNOS protein after 48 h (p<0.01,n=4). There was no difference between our preparation of ox-LDL andcommercially-available ox-LDL with similar TBARS values in terms of thedegree of ecNOS downregulation. Addition of simvastatin (0.01 mmol/L)did not significantly affect the downregulation of ecNOS protein byox-LDL (57%±8% decrease, p>0.05, n=4). However, in the presence of 0.1mmol/L of simvastatin, ox-LDL no longer produce any significant decreasein ecNOS protein levels (4% +7% decrease, p<0.01, n=4). Higherconcentrations of simvastatin (1 and 10 mmol/L) resulted in not only areversal of ecNOS downregulation by ox-LDL, but also significantincreases in ecNOS protein levels above baseline (146%±9% and 210% 112%,respectively, p<0.05, n=4). Simvastatin or lovastatin (10 mmol/L) whichwere not chemically-activated had no effect on ecNOS expression.

[0130] In a time-dependent manner, treatment with ox-LDL (50 mg/ml,TBARS 12.2 nmol/mg) decreased ecNOS protein expression by 34% (5%, 67%(8% and 86 (5% after 24 h, 72 h, and 96 h, respectively (p<0.05 for allvalues, n=4,). Compared to ox-LDL alone, co-treatment with simvastatin(0.1 mmol/L) attenuated the decrease in ecNOS protein level after 24 h(15% (2% vs 34% (5%, p<0.05, n=4). Longer incubation with simvastatin(0.1 mmol/L) for 72 h and 96 h not only reversed ox-LDL's inhibitoryeffects on ecNOS expression, but also increased ecNOS protein levels by110% (6% and 124% (6% above basal expression (p<0.05, n=4). Thus,compared to ox-LDL alone, co-treatment with simvastatin produced a 1.3-,3.3-and 8.9-fold increase ecNOS protein levels after 24 h, 72 h, and 96h, respectively.

Example 4 Effect of ox-LDL and HMG-CoA Reductase Inhibitors on ecNOSmRNA

[0131] The effect of simvastatin on ecNOS mRNA levels occurred in atime-dependent manner and correlated with its effect on ecNOS proteinlevels. Northern analyses showed that ox-LDL (50 mg/ml, TBARS 15.1nmol/mg) produced a time-dependent 65±5% and 91±4% decrease in ecNOSmRNA levels after 48 h and 72 h, respectively (p<0.01, n=3). Compared toox-LDL at the indicated time points, co-treatment with simvastatin 0.1mmol/L) increased ecNOS mRNA levels by 6.3-fold after 48 h and 14.5-foldafter 72 h (p<0.01 for all values, n=3).

[0132] To determine whether treatment with another HMG-CoA reductaseinhibitor have similar effect as simvastatin, we treated endothelialcells with lovastatin. Again, ox-LDL decreased steady-state ecNOS mRNAby 52±5% after 24 h (p<0.01, n=3). Treatment with lovastatin (10 mmol/L)not only reversed the inhibitory effects of ox-LDL on ecNOS mRNA, butalso caused a 40±9% increase in ecNOS mRNA level compared to that ofuntreated cells. Compared to ox-LDL alone, co-treatment with lovastatincaused a 3.6-fold increase in ecNOS mRNA levels after 24 h. Treatmentwith lovastatin alone, however, produced 36% increase in ecNOS mRNAlevels compared to untreated cells (p<0.05, n=3).

Example 5 Effect of ox-LDL and Simvastatin on ecNOS Activity

[0133] The activity of ecNOS was assessed by measuring theLNMA-inhibitable nitrite production from human endothelial cells (Liao,J K et al., J Clin Invest, 1995, 96:2661-2666). Basal ecNOS activity was8.8±1.4 nmol/500,000 cells/24 h. Treatment with ox-LDL (50 mg/ml, TBARS16 nmol/mg) for 48 h decreased ecNOS-dependent nitrite production by94±3% (0.6±0.5 nmol/500,000 cells/24 h, p<0.001). Co-treatment withsimvastatin (0.1 mmol/L) significantly attenuated this downregulationresulting in a 28±3% decrease in ecNOS activity compared to untreatedcells (6.4±0.3 nmol/500,000 cells/24 h, p<0.05). Co-treatment with ahigher concentration of simvastatin (1 mmol/L) not only completelyreversed the downregulation of ecNOS by ox-LDL, but also, resulted in a45±6% increase in ecNOS activity compared to baseline (12.8±2.7nmol/500,000 cells/24 h, p<0.05).

EXAMPLE 6 Effect of Simvastatin on ecNOS mRNA Stability

[0134] The post-transcriptional regulation of ecNOS mRNA was determinedin the presence of the transcriptional inhibitor, actinomycin D (5mg/ml). Oxidized LDL (50 mg/ml, TBARS 13.1 nmol/mg) shortened thehalf-life of ecNOS mRNA (t1/2 35±3 h to 14±2 h, p<0.05, n=3).Co-treatment with simvastatin (0.1 mmol/L) prolonged the half-life ofecNOS mRNA by 1.6-fold (t1/2 22±3 h, p<0.05, n=3). Treatment withsimvastatin alone prolonged ecNOS mRNA half-life by 1.3-fold overbaseline (t1/2 43±4 h, p<0.05, n=3).

EXAMPLE 7 Effect of Simvastatin on ecNOS Gene Transcription

[0135] To determine whether the effects of simvastatin on ecNOSexpression occurs at the level of ecNOS gene transcription, we performednuclear run-on assays using endothelial cells treated with simvastatin(1 mmol/L) for 24 h. Preliminary studies using different amounts ofradiolabelled RNA transcripts demonstrate that under our experimentalconditions, hybridization was linear and nonsaturable. The density ofeach ecNOS band was standardized to the density of its correspondingb-tubulin. The specificity of each band was determined by the lack ofhybridization to the nonspecific pGEM cDNA vector. In untreatedendothelial cells (control), there was constitutive ecNOStranscriptional activity (relative index of 1.0). Treatment withsimvastatin (1 mmol/L) did not significantly affect ecNOS genetranscription compared to that of untreated cells (relative index of1.2±0.3, p>0.05, n=4). However, treatment of endothelial cells withhyperoxia (95%_(O2)) significantly increased ecNOS gene expression(relative index of 2.5, p<0.05, n=4).

[0136] To further confirm the effects of simvastatin on ecNOS genetranscription by a different method, we transfected bovine aorticendothelial cells using a −1600 to +22 nucleotide ecNOS 5′-promoterconstruct linked to a luciferase reporter gene (F 1.LUC) (Zhang, R etal., J Biol Chem, 1995, 270:15320-15326). This promoter constructcontains putative cis-acting elements for activator protein (AP)-1 and-2, sterol regulatory element-1, retinoblastoma control element, shearstress response element (SSRE), nuclear factor-1 (NF-1), and cAMPresponse element (CRE). Treatment with ox-LDL (50 mg/ml, TBARS 14.5nmol/mg), simvastatin (1 μmol/L), alone or in combination, did notsignificantly affect basal F1 promoter activity. However, laminar fluidshear-stress (12 dynes/cm2 for 24 h) was able to induce F1 promoteractivity by 16-fold after 24 h (data not shown) indicating that the F1promoter construct is functionally-responsive if presented with theappropriate stimulus.

EXAMPLE 8 Effect of Simvastatin and Lovastatin on ecNOS Expression

[0137] To further characterize the effects of HMG-CoA reductaseinhibitors on the upregulation ecNOS expression, we treated endothelialcells with simvastatin (0.1 mmol/L) for various durations (0-84 h).Treatment with simvastatin (0.1 mmol/L) increased ecNOS protein levelsby 4 (6%, 21 (9%, 80 (8%, 90 (12%, and 95 (16% after 12 h, 24 h, 48 h,72 h, and 84 h, respectively (p<0.05 for all time points after 12 h,n=4). Higher concentrations of simvastatin similarly increased ecNOSprotein levels, but in significantly less time compared to lowerconcentrations of simvastatin (data not shown).

[0138] In a concentration-dependent manner, treatment with simvastatin(0.01 to 10 mmol/L, 48 h) increased ecNOS expression by 1 (6%, 80 (8%,190 (10% and 310 (20%, respectively (p<0.05 for concentrations (0.1mmol/L, n=4). The upregulation of ecNOS expression by simvastatin,therefore, is dependent upon both the concentration and duration ofsimvastatin treatment. For comparison, treatment with lovastatin (0.1 to10 mmol/L, 48 h) also increased ecNOS expression in aconcentration-dependant manner (10 (6%, 105 (8% and 180 (11%,respectively, p<0.05 for concentrations>0.1 mmol/L, n=3) butsignificantly less effectively than simvastatin at comparableconcentrations. Therefore, at the same concentration, simvastatin hadgreater effects on ecNOS expression compared to lovastatin. Theseresults are consistent with reported IC50 values for simvastatin andlovastatin (4 nmol/L and 19 nmol/L, respectively) (Van Vliet, A K etal., Biochem Pharmacol, 1996, 52:1387-1392).

EXAMPLE 9 Effect of L-Mevalonate on ecNOS Expression

[0139] To confirm that the effects of simvastatin on ecNOS expressionwere due to the inhibition of endothelial HMG CoA reductase, endothelialcells were treated with ox-LDL (50 mg/ml, TBARS 15.1 nmol/mg),simvastatin (1 mmol/L), alone or in combination, in the presence ofL-mevalonate (100 mmol/L). Treatment with ox-LDL decreased ecNOSexpression by 55% (6% after 48 h which was completely reversed andslightly upregulated in the presence of simvastatin (1 mmol/L) (150% (8%above basal expression) (p<0.05 for both, n=3).

[0140] Compared to endothelial cells treated with ox-LDL andsimvastatin, addition of L-mevalonate reduced ecNOS protein by 50%±5%(p<0.05, n=3). Furthermore, the upregulation of ecNOS expression bysimvastatin alone (2.9-fold increase, p<0.05, n=3) was completelyreversed by co-treatment with L-mevalonate. Treatment with L-mevalonatealone did not have any appreciable effects on basal ecNOS expression(p>0.05, n=3). Similar findings were also observed with L-mevalonate andlovastatin.

[0141] “HMG-CoA Reductase Inhibitors Reduce Cerebral Infarct Size byUpregulating endothelial cell Nitric Oxide Synthase”

[0142] Experimental Procedures

[0143] Cell Culture:

[0144] Human endothelial cells were harvested from saphenous veins usingType II collagenase (Worthington Biochemical Corp., Freehold, N.J.) aspreviously described. Cells of less than three passages were grown toconfluence in a culture medium containing Medium 199, 20 mM HEPES, 50mg/ml ECGS (Collaborative Research Inc., Bedford, Mass.), 100 mg/mlheparin sulfate, 5 mM L-glutamine (Gibco), 5% fetal calf serum (Hyclone,Logan, UT.), and antibiotic mixture of penicillin (100U/ml)/streptomycin (100 mg/ml)/Fungizone (1.25 mg/ml). For allexperiments, the endothelial cells were grown to confluence before anytreatment conditions. In some experiments, cells were pretreated withactinomycin D (5 mg/ml) for 1 h prior to treatment with HMG-CoAreductase inhibitors.

[0145] Exposure of Endothelial Cells to Hypoxia:

[0146] Confluent endothelial cells grown in 100 mm culture dishes weretreated with HMG-CoA reductase inhibitors and then placed withoutculture dish covers in humidified airtight incubation chambers(Billups-Rothenberg, Del Mar, Calif.). The chambers were gassed with 20%or 3% O₂, 5% CO₂, and balanced nitrogen for 10 min prior to sealing thechambers. The chambers were maintained in a 37° C. incubator for variousdurations (0-48 h) and found to have less than 2% variation in O₂concentration as previously described (Liao, J K et al., J Clin Invest,1995, 96:2661-2666). Cellular confluence and viability were determinedby cell count, morphology, and trypan blue exclusion.

[0147] In vitro Transcription Assay:

[0148] Confluent endothelial cells (5×10⁷ cells were treated withsimvastatin (1 mM) in the presence of 20% or 3% O₂ for 24 h. Nuclei wereisolated and in vitro transcription was performed as previouslydescribed (Liao, J K et al., J Clin Invest, 1995, 96:2661-2666). Equalamounts (1 mg) of purified, denatured full-length human ecNOS, humanb-tubulin (ATCC #37855), and linearized pGEM-3z cDNA werevacuum-transferred onto nitrocellulose membranes using a slot blotapparatus (Schleicher & Schuell). Hybridization of radiolabeled mRNAtranscripts to the nitrocellulose membranes was carried out at 45° C.for 48 h in a buffer containing 50% formamide, 5× SSC, 2.5× Denhardt'ssolution, 25 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250mg/ml salmon sperm DNA. The membranes were then washed with 1× SSC/0.1%SDS for 1 h at 65° C. prior to autoradiography for 72 h at −80° C. Bandintensities were subjected to analyses by laser densitometry.

[0149] Assay for Nitrite Accumulation:

[0150] The amount of NO produced by ecNOS was determined by nitriteaccumulation in the conditioned medium. Nitrite accumulation wasdetermined by measuring the conversion of 2,3-diaminonaphthalene (1.5 mMof DAN in 1 M of HCl) and nitrite to 1-(H)-naphthotriazole as previouslydescribed (13,24). Nonspecific fluorescence was determined in thepresence of LNMA (5 mM). Previous studies with nitrate reductaseindicate that the nitrite to nitrate concentration in the medium wasapproximately 5:1 and that this ratio did not vary with exposure to 20%or 3% O₂ concentration.

[0151] Murine Model of Cerebral Vascular Ischemia:

[0152] Adult male (18-20 g) wildtype SV-129 mice (Taconic farm,Germantown, N.Y.) and ecNOS mutant mice (Huang, PL et al., Nature, 1995,377:239-242.) were subcutaneously-injected with 0.2, 2, or 20 mg ofactivated simvastatin per kg body weight or saline (control) once dailyfor 14 days. Ischemia was produced by occluding the left middle cerebralartery (MCA) with a coated 8.0 nylon monofilament under anesthesia asdescribed (Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987,Huang, Z et al., Science, 1994, 265:1883-1885, Hara, H et al., J CerebBlood Flow Metab, 1997, 1:515-526). Arterial blood pressure, heart rate,arterial oxygen pressure, and partial pressure of carbon dioxide weremonitored as described (Huang, Z et al., J Cereb Blood Flow Metab, 1996,16:981-987, Huang, Z et al., Science, 1994, 265:1883-1885, Hara, H etal., J Cereb Blood Flow Metab, 1997, 1:515-526). The filaments werewithdrawn after 2 hours and after 24 h, mice were either sacrificed ortested for neurological deficits using a well-established, standardized,observer-blinded protocol as described (Huang, Z et al., J Cereb BloodFlow Metab, 1996, 16:981-987, Huang, Z et al., Science, 1994,265:1883-1885, Hara, H et al., J Cereb Blood Flow Metab, 1997,1:515-526). The motor deficit score range from 0 (no deficit) to 2(complete deficit).

[0153] Brains were divided into five coronal 2-mm sections using a mousebrain matrix (RBM-200C, Activated Systems, Ann Arbor, Mich., USA).Infarction volume was quantitated with an image analysis system (M4, St.Catharines, Ontario, Canada) on 2% 2,3,5-triphenyltetrazolium chloridestained 2-mm slices. The levels of serum cholesterol, creatinine andtransaminases were determined by the Tufts University VeterinaryDiagnostic Laboratory (Grafton, Mass.).

[0154] Assay for ecNOS Activity from Tissues:

[0155] The ecNOS activities in mice aortae and brains were measured bythe conversion of [³H]arginine to [³H]citrulline in the presence andabsence of LNMA (5 mM) as described earlier.

[0156] Ouantitative Reverse Transcription-Polymerase Chain Reaction:

[0157] Total RNA from mouse aortae and brains was isolated by theguanidinium isothiocyanate method and reverse transcribed using oligo-dT(mRNA Preamplification reagents; Gibco BRL) and Taq ploymerase(Perkin-Elmer). One tenth of the sDNA was used as template for the PCRreaction. Approximately 0.2 nmol of the following primers amplifying a254-bp fragment of murine ecNOS cDNA were used: 5′Primer:5′-GGGCTCCCTCCTTCCGGCTGCCACC-3′ (SEQ ID NO. 1) and 3′Primer:5′-GGATCCCTGGAAAAGGCGGTGAGG-3′ (SEQ ID NO. 2) (Hara, H et al., J CerebBlood Flow Metab, 1997, 1:515-526). For amplification of glyceraldehyde3-phosphate dehydrogenase (GAPDH), 0.1 nmol of the following primersamplifying a 452-bp fragment were used: 5′Primer:5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO. 3) and 3′ Primer:5′-TCCACCACCCTGTTGCTGTA-3′(SEQ ID NO. 4). Denaturing was performed at94° C. for 30 s, annealing at 60° C. for 30 s, and elongation at 72° C.for 60 s. Preliminary results indicated that the linear exponentialphase for ecNOS and GAPDH polymerization was 30-35 cycles and 20-25cycles, respectively.

EXAMPLE 10 Cell Culture

[0158] Relatively pure (>98%) human saphenous vein endothelial cellcultures were confirmed by their morphological features (ie. cuboidal,cobble-stone, contact inhibited) using phase-contrast microscopy andimmunofluorescent-staining with antibodies to Factor VIII. There were noobservable adverse effects of HMG-CoA reductase inhibitors, L-mevalonicacid, or hypoxia on cellular morphology. However, higher concentrationsof simvastatin (>15 mmol/L) or lovastatin (>50 mmol/L) causedcytotoxicity after 36 h, and therefore, were not used. Otherwise,cellular confluency and viability as determined by trypan blue exclusionwere maintained for all treatment conditions described.

Example 11 Effects of HMG-CoA Reductase Inhibitors on ecNOS Activity

[0159] The activity of ecNOS was assessed by measuring theLNMA-inhibitable nitrite accumulation from human endothelial cells(Liao, J K et al., J Clin Invest, 1995, 96:2661-2666). The ratio ofnitrite to nitrate production under our culture condition wasapproximately 5:1 and was similar for hypoxia and normoxia (data notshown). Basal ecNOS activity at 20% O₂ was 6.0±3.3 nmol/500,000 cells/24h. Exposure of endothelial cells to 3% O₂ for 24 h decreased nitriteproduction by 75±14% (1.5±0.9 nmol/500,000 cells/24 h, p<0.01).Treatment with simvastatin (1 mM) not only completely reversed thedownregulation of ecNOS by hypoxia, but resulted in a 3-fold increase inecNOS activity over basal activity (18±5.0 nmol/500,000 cells/24 h,p<0.05). This upregulation of ecNOS activity was attenuated by theaddition of L-mevalonate (400 mM) (9.6±1.3 nmol/500,000 cells/24 h,p<0.05). Interestingly, simvastatin (1 mM) alone upregulated nitriteproduction 5-fold (30±6.5 nmol/500,000 cells/24 h, p<0.01), which wascompletely blocked by L-mevalonate (400 mM) (8.6±2.9 nmol/500,000cells/24 h, p<0.05). Similar findings were observed with lovastatin, butat 10-fold higher concentration compared to that of simvastatin.

Example 12 Effects of HMG-CoA Reductase Inhibitors on ecNOS Protein andmRNA Levels

[0160] In a concentration-dependent manner, treatment with simvastatin(0.01 to 10 mM, 48 h) increased ecNOS expression by 1 (6%, 80 (8%, 190(10% and 310 (20%, respectively (p <0.05 for concentrations (0.1 mM,n=4). Treatment with simvastatin (0.1 mM) increased ecNOS protein levelsin a time-dependent manner by 4 (6%, 21 (9%, 80 (8%, 90 (12%, and 95(16% after 12 h, 24 h, 48 h, 72 h, and 84 h, respectively (p<0.05 forall time points after 12 h, n=4) (data not shown). Another HMG-CoAreductase inhibitor, lovastatin, also increased ecNOS protein levels ina time-, and concentration-dependent manner (data not shown). Becauselovastatin has a higher IC50 value for HMG-CoA reductase compared tothat of simvastatin, it was 10-fold less potent in upregulating ecNOSprotein levels than simvastatin at equimolar concentrations.

[0161] We have previously shown that hypoxia downregulates ecNOS proteinexpression (Liao, J K et al., J Clin Invest, 1995, 96:2661-2666).Compared to normoxia (20% O₂), exposure to hypoxia (3% O₂) resulted in a46±4% and 75±3% reduction in ecNOS protein levels after 24 h and 48 h,respectively (p<0.0 1, n=3). In a concentration-dependent manner,treatment with simvastatin produced a progressive reversal ofhypoxia-mediated downregulation of ecNOS protein levels after 48 h. Athigher concentrations of simvastatin (1 and 10 mM), ecNOS protein levelswere upregulated to 159±13% and 223±21% of basal levels (p<0.05, n=3).Co-treatment with L-mevalonic acid (400 mM) completely blockedsimvastatin-induced increase in ecNOS protein levels after 48 h(35±2.4%). Treatment with L-mevalonic acid alone, however, did notproduce any significant effects on basal ecNOS protein levels inuntreated cells exposed to hypoxia (25±3.9%, p>0.05, n=3). In addition,simvastatin which was not chemically-activated had no effect on ecNOSexpression. These results indicate that simvastatin- andlovastatin-mediated increases in ecNOS protein expression are mediatedby inhibition of endothelial HMG-CoA reductase.

[0162] To determine whether changes in ecNOS protein levels are due tochanges in ecNOS steady-state mRNA levels, we performed Northernblotting on endothelial cells exposed to normoxia and hypoxia in thepresence or absence of simvastatin (1 mM) and lovastatin (10 μM).Simvastatin alone increased ecNOS mRNA levels to 340±24% (p<0.01, n=3).Exposure of endothelial cells to hypoxia reduced ecNOS mRNA levels by70%±2% and 88±4% after 24 h and 48 h with respect to GAPDH mRNA levels,respectively. Co-treatment with simvastatin not only completely reversedhypoxia-mediated decrease in ecNOS mRNA levels, but increased ecNOS mRNAlevels to 195±12% and 530±30% of basal levels after 24 h and 48 h,respectively (p<0.01, n=3). Similarly, lovastatin (10 μM) aloneincreased ecNOS message to 350±27% under hypoxia and 410±21% alone(p<0.01, n=3). Neither simvastatin nor lovastatin caused any significantchange in G-protein as and b-actin mRNA levels under normoxic or hypoxicconditions. These results indicate that the effects of HMG-CoA reductaseinhibitors are relatively selective in terms of their effects on ecNOSmRNA expression.

Example 13 Effects of HMG-CoA Reductase Inhibitors on ecNOS mRNAHalf-life

[0163] The half-life of ecNOS mRNA was determined in the presence ofactinomycin D (5 mg/ml). Hypoxia shortened the half-life of ecNOS mRNAfrom 28±4 h to 13±3 h. Treatment with simvastatin (1 mM) increased ecNOShalf-life to 46±4 h and 38±4 h under normoxic and hypoxic conditions,respectively (p<0.05 for both, n=3). These results suggest that HMG-CoAreductase inhibitors prevent hypoxia-mediated decrease in ecNOSexpression by stabilizing ecNOS mRNA.

EXAMPLE 14 Effects of HMG-CoA Reductase Inhibitors on ecNOS GeneTranscription

[0164] Nuclear run-on assays showed that hypoxia caused a 85±8% decreasein ecNOS gene transcription (p<0.01, n=3). Treatment with simvastatin (1mM) did not produce any significant affect on hypoxia-mediated decreasein ecNOS gene transcription (83±6% decrease in ecNOS gene transcription,p>0.05 compared to hypoxia alone). Furthermore, simvastatin aloneproduced minimal increase in ecNOS gene transcription under normoxiccondition (20±5% increase in ecNOS gene transcription, p<0.05 comparedto normoxia control).

[0165] Preliminary studies using different amounts of radiolabeled RNAtranscripts demonstrate that under our experimental conditions,hybridization was linear and nonsaturable. The density of each ecNOSband was standardized to the density of its corresponding (b-tubulinband, relative intensity). To exclude the possibility that changes in(b-tubulin gene transcription are caused by hypoxia or simvastatin,another gene, GAPDH, was included on each of the nuclear run-on blots.Similar relative indices were obtained when ecNOS gene transcription wasstandardized to GAPDH gene transcription. The specificity of each bandwas determined by the lack of hybridization to the nonspecific pGEM cDNAvector.

EXAMPLE 15 Effect of HMG-CoA Reductase Inhibitors on Mouse Physiology

[0166] To determine whether the upregulation of ecNOS by HMG-CoAreductase inhibitors occurs in vivo, SV-129 wild-type and ecNOS knockoutmice were treated with 2 mg/kg simvastatin or saline subcutaneously for14 days (n=8). The mean arterial blood pressures of wild-type and ecNOSmutant mice were as reported previously (Huang, PL et al., Nature, 1995,377:239-242). The ecNOS mutants were relatively hypertensive. There wasno significant change in mean arterial blood pressures of wild-type miceafter 14 days of simvastatin treatment (81 +7 mmHg vs. 93±10 mmHg,p>0.05). There was also no significant group difference in heart rate,arterial blood gases and temporalis muscle temperature before ischemiaor after reperfusion. Furthermore, there was no significant differencein the levels of serum cholesterol (control: 147±10 vs. simvastatin161±5.2 mg/dl), creatinine and transaminases after treatment withsimvastatin compared to control values.

EXAMPLE 16 Effect of HMG-CoA Reductase Inhibitors on ecNOS Expressionand Function in Mouse Aorta

[0167] The activity of ecNOS in the aortae of simvastatin-treated (2mg/kg) and saline-injected mice was determined by measuring theLNMA-inhibitable conversion of arginine to citrulline (FIG. 1A). TheecNOS activity in aortae from simvastatin-treated mice was significantlyhigher than in the control group (0.39±0.09 vs. 0.18±0.04 U/mg protein,n=8, p<0.05).

[0168] The ecNOS mRNA expression in the aortae of simvastatin-treatedand -untreated mice was examined by quantitative RT-PCR (FIG. 1B). Therewas a significantly dose-dependent 3-fold increase of ecNOS messagecompared to that of GAPDH in simvastatin-treated mice (n=3). Thesefindings indicate that simvastatin upregulates ecNOS expression andactivity in vivo.

EXAMPLE 17 Effect of HMG-CoA Reductase Inhibitors on Cerebral Ischemiain Mice

[0169] Endothelium-derived NO protects against ischemic cerebral injury(Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987). Thereforewe examined, wether the observed upregulation of ecNOS by simvastatin invivo has beneficial effects on cerebral infarct size. Followingtreatment for 14 days with 2 mg/kg of simvastatin, cerebral ischemia wasproduced by occluding the left middle cerebral artery for 2 hours. After22 hours of reperfusion, mice were tested for neurological deficitsusing a well-established, standardized, observer-blinded protocol. Theneurological motor deficit score improved in simvastatin-treated mice(n=18) by almost 2-fold compared to that of controls (n=12) (0.8±0.2 vs.1.7±0.2, p<0.01).

[0170] Simvastatin-treated wild-type mice (n=18) had 25% smallercerebral infarct sizes compared to untreated animals (73.8±8.5 mm3 vs.100.7±7.3 mm3, n=12, p<0.05). This effect was concentration-dependent(0.2, 2, 20 mg/kg simvastatin), persisted for up to 3 days, and alsooccurred with lovastatin treatment, albeit at higher relativeconcentrations (data not shown). Furthermore, simvastatin increasecerebral blood flow by 23% and 35% over basal values at concentrationsof 2 mg/kg and 20 mg/kg, respectively (n=8, p<0.05 for both). Thesefindings suggest, that simvastatin decreases cerebral infarct size andneurological deficits.

[0171] Finally, to demonstrate that the reduction of cerebral infarctsizes by simvastatin is due to the upregulation of ecNOS, cerebralischemia was applied to ecNOS mutant mice lacking ecNOS gene in thepresence and absence of simvastatin (2 mg/kg, 14 days). There was nosignificant difference between the cerebral infarct sizes ofsimvastatin-treated and -untreated ecNOS mutant mice (n=6, p<0.05).These findings indicate that the upregulation of ecNOS mediates thebeneficial effects of HMG-CoA reductase inhibitors on cerebral infarctsize.

Example 18 Effect of HMG-CoA Reductase Inhibitors on ecNOS Expression inMouse Brain

[0172] The ecNOS mRNA expression in the ischemic and contralateral(non-ischemic) hemispheres of mouse brain was examined by quantitativeRT-PCR (FIG. 2) with respect to GAPDH mRNA levels. Simvastatin-treatedmice (n=3) (2 mg/kg, 14 days) showed a 1.5- to 2-fold increase in ecNOSexpression in the infarcted, ipsilateral hemisphere compared to thecontralateral, non-infarcted side. In contrast, there was no differencein ecNOS expression in untreated mice between their infarcted andnon-infacted hemispheres. These findings suggest that simvastatin mayreduced cerebral infarct size by selectively increasing ecNOS expressionin the ischemic and hypoxic infarct zone.

[0173] “Regulation of Endothelial Nitric Oxide Synthase Expression byRho GTPases”

[0174] Experimental Procedures

[0175] Materials:

[0176] Mevastatin, farnesylpyrophosphate, geranylgeranylpyrophosphate,and L-mevalonate were purchased from Sigma Chemical Corp. (St. Louis,Mo.). Mevastatin (compactin- a HMG-CoA reductase inhibitor) waschemically activated by alkaline hydrolysis prior to use as previouslydescribed (Laufs, U et al., J Biol Chem, 1997, 272:31725-31729). FPTinhibitor I and -hydroxyfamesylphosphonic acid were purchased fromCalbiochem Corp. (La Jolla, Calif.). [a-³²P]CTP (3000 Ci/mmol) and [³⁵S]GTP S (1250 Ci/mmol) were supplied by New England Nuclear. Theantibody detection kit (Enhanced Chemiluminescence) and the nylonnucleic acid (Hybond) and protein (PVDF) transfer membranes werepurchased from Amersham Corp. (Arlington Heights, Ill.). The Clostridiumbotulinum C3 transferase was purchased from List BiologicalLaboratories, Inc. (Campbell, Calif.). Recombinant Escherichia colicytotoxic necrotizing factor (CNF)-1 and RhoA mutants were kindlyprovided by K. Aktories (University of Freiberg, Germany) and W.Moolenaar (Netherlands Cancer Institute, Netherlands), respectively.

[0177] Cell Culture

[0178] Human endothelial cells were harvested using Type II collagenase(Worthington Biochemical Corp., Freehold, N.J.) as previously described(Laufs, U et al., J Biol Chem, 1997, 272:31725-31729; Liao, J K et al.,J Biol Chem, 1995, 270:319-324). Cells of less than three passages weregrown in a culture medium containing Medium 199, 20 mM HEPES, 50 mg/mlECGS (Collaborative Research Inc., Bedford, Mass.), 100 mg/ml heparinsulfate, 5 mM L-glutamine (Gibco), 5% fetal calf serum (Hyclone, Logan,Utah), and antibiotic mixture of penicillin (100 U/ml)/streptomycin (100mg/ml)/Fungizone (1.25 mg/ml). Confluent endothelial cells were used forall treatment conditions. For transfection studies, bovine aorticendothelial cells of less than 3 passages were cultured in a growthmedium containing DMEM (Dulbecco's Modified Eagle's Medium), 5 mM ofL-glutamine (Gibco), and 10% fetal calf serum. Cellular viability wasdetermined by cell count, morphology, and trypan blue exclusion.

[0179] Preparation of LDL

[0180] The LDL was prepared as described earlier. The extent of LDLoxidation was estimated by assaying for thiobarbituric acid reactivesubstances (TBARS) and expressed as nanomoles of malondialdehyde per mgof LDL protein, as described earlier. Only freshly-isolated LDL withTBARS values of less than 0.5 nmol/mg was used in this study.

[0181] Western Blotting

[0182] Proteins were prepared and separated on SDS/PAGE as describedearlier. Immuno-blotting was performed using monoclonal antibodies toecNOS (1:400 dilution, Transduction Laboratories, Lexington, Ky.), toRhoA and RhoB (1:250 dilution, Santa Cruz Biotechnology Inc., SantaCruz, Calif.), and to c-myc-tag (9E10, 1:200 dilution, Santa CruzBiotechnology Inc.). Immunodetection was accomplished using a sheepanti-mouse secondary antibody (1:4000 dilution) or donkey anti-rabbitsecondary antibody (1:4000 dilution) and the enhanced chemiluminescence(ECL) kit (Amersham Corp., Arlington Heights, Ill.). Autoradiography wasperformed as described earlier.

[0183] Assay for Rho GTP-binding Activity

[0184] The Rho GTP-binding activity was determined byimmunoprecipitation of [³⁵ S]GTP S-labeled Rho. Briefly, membrane andcytosolic proteins were isolated as previously desribed (Liao, J K andHomcy, C J, J Clin Invest, 1993, 92:2168-2172). Proteins (20 mg) fromcontrol and treated endothelial cells were incubated for 30 min at 37°C. in a buffer containing [35 S]GTP S (20 nM), GTP (2 mM), MgCl₂₂ (5mM), EGTA (0.1 mM), NaCl (50 mM), creatinine phosphate (4 mM),phosphocreatinine kinase (5 units), ATP (0.1 mM), dithiothreitol (1 mM),leupeptin (100 mg/ml), aprotinin (50 mg/ml), and phenylmethanesulfonylfluoride (PMSF, 2 mM). The assay was terminated with excess unlabeledGTP S (100 mM).

[0185] Samples were then resuspended in 100 ml of immunopricipitationbuffer containing Triton-X (1%), SDS (0.1%), NaCl (150 mM), EDTA (5 mM),Tris-HCl (25 mM, pH 7.4), leupeptin (10 mg/ml), aprotinin (10 mg/ml),and PMSF (2 mM). The RhoA or RhoB antisera were added to the mixture ata final dilution of 1:75. The samples were allowed to incubate for 16 hat 4° C. with gentle mixing. The antibody-G-protein complexes were thenincubated with 50 ml of protein A-Sepharose (1 mg/ml, Pharmacia BiotechInc.) for 2 h at 4° C., and the immuno-precipitate was collected bycentrifugation at 12,000× g for 10 min.

[0186] Preliminary studies using Western analysis of the supernatantindicated that both RhoA and RhoB were completely immunoprecipitatedunder these conditions. The pellets were washed four times in a buffercontaining HEPES (50 mM, pH 7.4), NaF (100 mM), sodium phosphate (50mM), NaCl (100 mM), Triton X-100 (1%), and SDS (0.1%). The final pelletcontaining the immunoprecipitated [35 S]GTP S-labeled Rho proteins wascounted in a liquid scintillation counter (LS 1800, Beckman Instruments,Inc. Fullerton, Calif.). Nonspecific activity was determined in thepresence of excess unlabeled GTP S (100 mM).

[0187] Overexpression of Rho Mutants

[0188] For transfection studies, bovine rather than human endothelialcells were used because of their higher transfection efficiency by thecalcium-phosphate precipitation method (12% vs <4%) (15). Bovineendothelial cells (60-70% confluent) were transfected with 15 mg of theindicated cDNAs: the insertless vector (pcDNA3), pcDNA3-c-myc-wtRhoA(wildtype RhoA), and pcDNA3-c-myc-N 19RhoA (dominant-negative RhoAmutant) (Gebbink, M et al., J Cell Biol, 1997, 137:1603-1613). As aninternal control for transfection efficiency, pCMV. b-Gal plasmid (5 mg)was co-transfected. Preliminary results using b-galactosidase stainingindicate that cellular transfection efficiency was approximately 10% to14%. The b-galactosidase activity was determined by a chemiluminescenceassay (Dual-Light, Tropix, Bedford, Mass.) using a Berthold L9501luminometer. Approximately 24 h after transfection, cells were harvestedfor immunoblot analyses of ecNOS expression. The ecNOS protein levelswere then standardized to the corresponding levels of transfected RhoAexpression as determined by antisera to the corresponding c-myc tag.

[0189] Assay for ecNOS Activity

[0190] The ecNOS activity was determined by a modified nitrite assay aspreviously described. Briefly, endothelial cells grown in phenol-freemedium were exposed to C3 transferase (50 mg/ml), FPP (10 mM), GGPP (5mM), CNF-1 (200 ng/ml), or mevastatin (10 mM). After 24 h, conditionedmedium (300 ml) was mixed with 30 ml of freshly-prepared2,3-diaminonaphthalene (1.5 mM of DAN in 1 M of HCI). The mixture wasprotected from light and incubated at 20° C. for 10 min. The reactionwas terminated with 15 ml of 2.8 M of NaOH. Fluorescence of1-(H)-naphthotriazole was measured with excitation and emissionwavelengths of 365 and 450 nm, respectively. Standard curves wereconstructed with known amounts of sodium nitrite. Nonspecificfluorescence was determined in the presence of LNMA (3 mM). Previousstudies with nitrate reductase indicate that the nitrite to nitrateconcentration in the medium was approximately 5:1 and that this ratiodid not vary under the described treatment conditions (Laufs, U et al.,J Biol Chem, 1997, 272:31725-31729).

[0191] Data Analysis

[0192] Band intensities from Northern and Western blots were analyzed asdescribed earlier. Paired and unpaired Student's t-tests were employedto determine the significance of changes in densitometric measurements,GTP-binding activities, and nitrite levels. A significant difference wastaken for p<0.05.

EXAMPLE 19 Cell Culture

[0193] Relatively pure (>98%) human saphenous vein endothelial cellcultures were confirmed by their morphological features (i.e. cuboidal,cobble-stone, contact inhibited) using phase-contrast microscopy andimmunofluorescent-staining with antibodies to Factor VIII (data notshown). There were no observable adverse effects of mevastatin, FPP,GGPP, C3 transferase, and CNF-1 on cellular viability. However, higherconcentrations of mevastatin (>50 mM) or CNF-1 (>5 mg/ml) did producecytotoxicity and therefore were not used. Cellular confluency andviability as determined by light microscopy and trypan blue exclusionwere maintained for all treatment conditions described.

Example 20 Effects of Isoprenoid Intermediates on ecNOS mRNA Expression

[0194] We have shown that inhibition of endothelial HMG-CoA reductase bylovastatin or simvastatin upregulates ecNOS expression and activity viaincreases in ecNOS mRNA stability (Examples 3-8 and Laufs, U et al., JBiol Chem, 1997, 272:31725-31729). Similarly, treatment of endothelialcells with mevastatin (10 mM) increased ecNOS steady-state mRNA levelsby 405±15% after 24 h (FIG. 3A). On a molar basis, we find thatmevastatin is equally potent compared with lovastatin but approximatelyten times less potent compared to simvastatin. This is consistent withtheir relative IC₅₀ values for HMG-CoA reductase inhibition (Blum, CB,Am. J. Cardiol, 1994, 73:3D-11D).

[0195] To determine which downstream isoprenoid intermediate in thecholesterol biosynthetic pathway regulates ecNOS expression, endothelialcells were treated with mevastatin (10 mM) in the presence or absence ofisoprenoid intermediates, geranylgeranylpyrophosphate (GGPP) orfamesylpyrophosphate (FPP). Co-treatment with FPP (10 mM) mildly reducedecNOS mRNA levels compared to mevastatin alone. However, co-treatmentwith GGPP (10 mM) completely reversed the upregulation of ecNOS mRNAlevels by mevastatin. In a concentration-dependent manner, GGPP reversedthe effects of mevastatin (10 mM) with complete reversal occuring at aGGPP concentration of 5 mM (FIG. 3B). Interestingly, treatment with GGPP(10 mM) alone did not significantly affect basal ecNOS mRNA levels.

[0196] Similarly, treatment with mevastatin (10 mM) increased ecNOSprotein levels by 180±11% after 24 h (p<0.05, n=4) (FIG. 4).Co-treatment with FPP (10 mM) or LDL (1 mg/ml) did not significantlyreverse the effects of mevastatin on ecNOS protein levels. Furthermore,inhibition of protein farnesyltransferase with the farnesyl-proteintransferase inhibitor I (0.5-50 mM) or -hydroxyfarnesylphosphonic acid(2-20 mM) did not affect ecNOS protein levels. In contrast, co-treatmentwith GGPP at a concentration of 10 mM, but not 1 mM, completely reversedthe upregulation of ecNOS protein levels by mevastatin. These findingsindicate that ecNOS expression is negatively regulated bygeranylgeraniol synthesis.

EXAMPLE 21 Effects of Mevastatin on Rho Membrane Translocation

[0197] The geranylgeranylation of the small GTPases, RhoA and RhoB, areessential for their membrane translocation from the cytosol (Van Aelst,L and D'Souza-Schorey, C, Genes Dev, 1997, 11:2295-2322). Under basalculture conditions, both RhoA and RhoB are present in the membranes andcytosol. Treatment with mevastatin decreased membrane localization ofRhoA and RhoB by 60±5% and 78±6% and produce a concomitant increase inRhoA and RhoB in the cytosol by 65±4% and 87±7%. Co-treatment with GGPP(5 mM), but not FPP (10 mM) reversed the effects of mevastatin andcompletely restored the amount of cytosolic and membrane-asociated RhoAand RhoB to basal levels. These findings suggest that inhibition of Rhogeranylgeranylation by mevastatin prevents RhoA and RhoB fromtranslocating to and associating with the cellular membrane.

Example 22 Effects of Mevastatin on Rho GTP-Binding Activity

[0198] To determine whether geranylgeranylation of RhoA and RhoB affectstheir activity (i.e. GTP-bound state), we immunoprecipitated [³⁵S]GTPS-labeled RhoA and RhoB from the membrane and cytosol of endothelialcells treated with mevastatin (10 mM) in the presence of GGPP (5 mM) orFPP (10 mM). Under basal conditions, endothelial cells havemembrane-associated RhoA and RhoB activity of 4.4±0.1 fmol/mg/min and3.8+0.4 fmol/mg/min, respectively. Treatment with mevastatin decreasedmembrane-associated RhoA and RhoB GTP-binding activity by 52% (2.1±0.4fmol/mg/min; p<0.01) and 37% (2.4±0.6 fmol/mg/min; p<0.05), respectively(n=3).

[0199] Co-treatment with FPP (10 mM) produced no significant effects onRhoA and RhoB GTP-binding activity compared to mevastatin alone (2.6±0.9fmol/mg/min and 2.7±0.5 fmol/mg/min, respectively, p>0.05, n=3).However, co-treatment with GGPP (10 mM) completely reversed theinhibitory effects of mevastatin on RhoA and RhoB GTP-binding activity(4.1±0.3 fmol/mg/min and 3.6±0.5 fmol/mg/min, respectively, p<0.05,n=3). Cytosolic RhoA and RhoB were relatively inactive (i.e. <1fmol/mg/min) and their activities were not affected by treatment withmevastatin alone or in combination with GGPP or FPP. Taken together,these results indicate that geranylgeranylation of RhoA and RhoB isnecessary for their membrane translocation and that membrane-associatedRho is relatively more active in terms of GTP-binding than cytosolicRho.

EXAMPLE 23 Effects of C3 Transferase on ecNOS Expression

[0200] To determine whether the inhibition of Rho mediates the effectsof mevastatin on ecNOS expression, endothelial cells were treated withmevastatin in the presence and absence of Clostridium botulinum C3transferase (5-50 mg/ml), an exoenzyme which specifically inactivatesRho by ADP-ribosylation (Aktories, K, J Clin Invest, 1997, 12:S11-S13).Treatment of endothelial cells with mevastatin (10 mM) or C3 transferase(50 mg/ml) for 48 h augmented ecNOS protein levels by 260±9% and250±10%, respectively (p<0.01, n=3). Lower concentrations of C3transferase (i.e. <50 mg/ml) produced correspondingly smaller increasesin ecNOS expression (data not shown). In contrast to the effect ofmevastatin, the stimulatory effect of C3 transferase on ecNOS expressionwas not reversed in the presence of L-mevalonate (200 mM).

EXAMPLE 24 Effects of Dominant-Negative RhoA on ecNOS Expression

[0201] Bovine aortic endothelial cells were transfected with insertlesspcDNA3 vector, c-myc-tagged wildtype RhoA (wtRhoA), or c-myc-taggeddominant-negative RhoA mutant (N19RhoA) which cannot exchange GDP forGTP and therefore is inactive (Gebbink, M et al., J Cell Biol, 1997,137:1603-1613). Immunostaining for b-galactosidase activity demonstratecomparable transfection efficiency of approximately 10% among the RhoAconstructs and between treatment conditions. To distinguish betweentransfected and endogenous RhoA, the amount of transfected RhoAconstructs expressed was assessed by immunoblotting using an antibody toc-myc (9E 10), which recognizes a 21 kD band only in wtRhoA and N19RhoAtransfected cells (FIG. 6).

[0202] Overexpression of wtRhoA mildly reduced basal ecNOS proteinexpression by 15±4% suggesting that increased RhoA expression results ina decrease in basal ecNOS expression (p<0.05, n=3). Endothelial cellstransfected with the dominant-negative N19RhoA mutant to comparablelevels as wtRhoA as assessed by the amount of c-myc-tag, however,exhibited a 150±5% increase in ecNOS protein levels (p<0.05, n=3). Theobserved effects of N19RhoA overexpression on overall ecNOS proteinlevels (i.e. transfected and non-transfected cells) are more profoundwhen one considers that only 10% of the endothelial cells were actuallytransfected. These findings are consistent with our earlier findingsthat inhibition of Rho GTPase activity leads to an increase in ecNOSexpression.

EXAMPLE 25 Effects of CNF-1 on ecNOS Expression

[0203] The Escherichia coli cytotoxic necrotizing factor (CNF)-1 isknown to directly and specifically activate rho proteins via glutaminedeamination (Aktories, K, J Clin Invest, 1997, 12:S11-S13; Schmidt, G etal., Nature, 1997, 387:725-729; Flatau, G et al., Nature, 1997,387:729-733). Treatment of endothelial cells with mevastatin (10 mM)increased ecNOS mRNA levels by 390±15% compared to basal levels (p<0.01,n=3). Co-treatment with CNF-1 (200 ng/ml) completely reversed theupregulation of ecNOS mRNA by mevastatin (p>0.05 compared to basallevels, n=3). Treatment with CNF-1 (200 ng/ml) alone, however, decreasedecNOS steady-state mRNA levels to 48±6% of basal levels at 24 h (p<0.05,n=3). These findings indicate that the direct activation of Rho leads tothe downregulation of ecNOS expression.

EXAMPLE 26 Effects of HMG-CoA Reductase Inhibitors and Rho on ecNOSactivity

[0204] The ecNOS activity was assessed by measuring the LNMA-inhibitablenitrite accumulation in conditioned media of endothelial cells (Laufs, Uet al., J Biol Chem, 1997, 272:31725-31729). Basal ecNOS activity was9.7±1.4 nmol/500,000 cells/24 hours (FIG. 7). Treatment of endothelialcells with mevastatin (10 mM) resulted in a 3-fold increase in nitriteaccumulation (32±1.9 nmol/500,000 cells/24 hours, p<0.01). This increasein ecNOS activity by mevastatin was reversed by co-treatment with GGPP(5 mM), but not FPP (10 mM) (12±0.8 and 27±4.9 nmol/500,000 cells/24hours, respectively). Furthermore, direct activation of Rho by CNF-1(200 ng/ml) reversed mevastatin-induced increase in ecNOS activity(32±1.9 to 14±2.1 nmol/500,000 cells/24 hours, p<0.05). In contrast,inhibition of Rho by C3 transferase (50 mg/ml) resulted in a 3-foldincrease in nitrite accumulation (31±2.1 and ±nmol/500,000 cells/24hours, p<0.05). These results indicate that Rho not only negativelyregulates ecNOS expression, but also ecNOS activity.

[0205] “Regulation of Endothelial Nitric Oxide Synthase Activity byAgents that Disrupt Actin Cytoskeletal Organization”

[0206] Experimental Procedures

[0207] Materials:

[0208] Myosin light chain kinase inhibitors BDM [2,3-butanedione2-monoxime], ML-7[1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride], and H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazinedihydro-chloride], were purchased from Sigma Chemical Corp. (St. Louis,Mo.). Nocodazole

[0209] {Methyl-(5-[2-thienylcarbonyl]-1H-benzimidazol -2-yl)carbamate}was also purchased from Sigma.

[0210] Cell culture, Western blotting and Northern blotting wereperformed as described earlier.

EXAMPLE 27 Effects of Agents That Disrupt Actin CytoskeletalOrganization on ecNOS Protein and mRNA Expression

[0211] To determine whether downstream targets of rhoGTPase exert anyeffect on ecNOS expression, endothelial cells were treated in thepresence and absence of a myosin light chain (MLC) kinase inhibitor, forexample, H-7. MLC kinase inhibitors decrease MLC phosphorylation andstress fiber formation. Treatment of endothelial cells with H-7 (1-100mM) for 24 hours augmented ecNOS protein levels (FIG. 8). Similarexperiments using a different MLC kinase inhibitor (ML-7), producedidentical results.

[0212] Furthermore, disruption of the actin cytoskeletal organization byCytochalasin D (an agent that interferes with actin polymerization) orBDM (a different MLC kinase inhibitor), also lead to upregulation ofecNOS (FIGS. 9 and 10 respectively).

[0213] Inhibition, however, of cell cycle progression by anaphicoline(data not shown) or disruption of the microtubular cytoskeleton byenhancing microtubule depolymerization with nocodazole, do not increaseecNOS expression (FIG. 11).

[0214] These findings show that the uregulation of ecNOS expression isrelatively specific to disruption of the actin cytoskeleton.Additionally, the increased ecNOS expression by Cytochalasin D or BDMdoes not result from nonspecific cytotoxicity of these agents.

DETAILED DESCRIPTION OF THE DRAWINGS

[0215]FIG. 1. A) NOS-Activity measured by (C14)-arginine-citrullineassay in the aortas of wild-type SV-129 mice after treatment withsimvastatin (Sim, 2 mg/kg, s.c., 14 days) and of mice injected with PBS(Control), n=8, p<0.05.

[0216] B) ecNOS mRNA expression determined by quantitative polymerasechain reaction in wild-type SV-129 mice aortas after treatment withsimvastatin (Sim0.2, 0.2 mg/kg, s.c. and Sim20, 20 mg/kg, s.c.) for 14days and of mice injected with saline (Control) in comparison toglyceraldehyde 3-phosphate dehydrogenase (G3DPH) mRNA expression.

[0217] ecNOS expression and function is upregulated in the aortas ofmice treated with Sim.

[0218]FIG. 2. ecNOS mRNA expression in the infarcted, ipsolateral (I)and not-infarcted, contralateral (C) forebrain hemispheres of SV-129mice after treatment with simvastatin (Sim, 2 mg/kg, s.c., 14 days) andmice injected with saline (Control), as determined by quantitativepolymerase chain reaction compared to glyceraldehyde 3-phosphatedehydrogenase (G3DPH) mRNA expression. ecNOS mRNA expression wasupregulated in the infarcted brain area in Sim-treated animals.

[0219]FIG. 3. A) Northern analyses (20 mg total RNA/lane) showing theeffects of mevastatin (Statin, 10 mM) alone or in combination with FPP(10 mM) or GGPP (10 mM) on eNOS steady-state mRNA levels at 24 h. B)Concentration-dependent effects of GGPP (1-10 mM) on mevastatin (10mM)-induced increases in eNOS mRNA levels after 24 h. Each experimentwas performed three times with comparable results. The correspondingethidium bromide-stained 28S band intensities were used to standardizeloading conditions.

[0220]FIG. 4. Immunoblots (30 mg protein/lane) showing the effects ofmevastatin (Statin, 10 mM) alone or in combination with FPP (10 mM),GGPP (1-10 mM), or LDL cholesterol (LDL-C, 1 mg/ml) on eNOS proteinlevels after 24 h. The blot is representative of three separateexperiments.

[0221]FIG. 5. Immunoblot (30 mg protein/lane) showing the effects of C3transferase (C3, 50 mg/ml), mevastatin (Statin, 10 mM), or L-mevalonate(Mev, 200 mM) on eNOS protein levels after 48 h. The blot isrepresentative of three separate experiments.

[0222]FIG. 6. Immunoblots (30 mg protein/lane) showing eNOS proteinlevels after transfection with insertless vector, pcDNA3 (C),c-myc-wildtype-RhoA (wt), and c-myc-N19RhoA (dominant-negative rhoAmutant). The levels of overexpressed RhoA mutants were determined byimmunoblotting for their corresponding c-myc-tags (c-myc-RhoA).Experiments were performed three times with similar results.

[0223]FIG. 7. Effects of C3 transferase (C3, 50 mg/ml), FPP (10 mM),GGPP (5 mM), and CNF-1 (200 ng/ml) on mevastatin (Statin, 10 mM)-inducedeNOS activity as determined by LNMA-inhibitable nitrite production at 24h. Experiments were performed three times in duplicate with less than 5%variation. *p<0.05 compared with control (C), **p<0.05 compared withmevastatin.

[0224]FIG. 8. Immunoblots (30 mg protein/lane) showing theconcentration-dependent effects of MLC kinase inhibitor H-7on ecNOSprotein levels after 24 hours.

[0225]FIG. 9. Northern blot analysis (20 mg total RNA/lane) showingecNOS mRNA expression of endothelial cells treated with cytochalasin Dat 24 hours.

[0226]FIG. 10. Immunoblots (30 mg protein/lane) showing theconcentration-dependent effects of 2, 3-butanedione 2-monoxime on ecNOSprotein levels.

[0227]FIG. 11. Northern blot analysis (20 mg total RNA/lane) showingecNOS mRNA expression of endothelial cells treated with nocodazole for24 hours.

[0228] Equivalents

[0229] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

[0230] A Sequence Listing follows the claims.

[0231] All references disclosed herein are incorporated by reference intheir entirety.

1 4 1 25 DNA Mus musculus 1 gggctccctc cttccggctg ccacc 25 2 24 DNA Musmusculus 2 ggatccctgg aaaaggcggt gagg 24 3 20 DNA Mus musculus 3accacagtcc atgccatcac 20 4 20 DNA Mus musculus 4 tccaccaccc tgttgctgta20

We claim:
 1. A method for treating a hypoxia-induced condition in asubject, comprising: administering to a subject who has an abnormallyelevated risk of developing a hypoxia-induced condition or who has ahypoxia-induced condition an agent that disrupts actin cytoskeletalorganization in an amount effective to increase endothelial cell NitricOxide Synthase activity in the subject, provided that the agent thatdisrupts actin cytoskeletal organization is not a rho GTPase functioninhibitor.
 2. The method of claim 1, wherein the subject isnonhyperlipidemic.
 3. The method of claim 1, wherein the agent thatdisrupts actin cytoskeletal organization is administeredprophylactically to a subject who has an abnormally elevated risk ofdeveloping a hypoxia-induced condition.
 4. The method of claim 1,wherein the agent that disrupts actin cytoskeletal organization isadministered to a subject who has a hypoxia-induced condition.
 5. Themethod of claim 1, wherein the subject has an impaired lung function. 6.The method according to any one of claims 1-5, wherein the agent thatdisrupts actin cytoskeletal organization is selected from the groupconsisting of a myosin light chain kinase inhibitor, a myosin lightchain phosphatase, a protein kinase N inhibitor, a phospatidylinositol4-phosphate 5-kinase inhibitor, and cytochalasin D.
 7. The method ofclaim 6, wherein the myosin light chain kinase inhibitor is selectedfrom the group consisting of 2,3-butanedione 2-monoxime,1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1 ,4-diazepinehydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazinedihydro-chloride.
 8. The method according to any one of claims 1-5,further comprising co-administering a substrate of endothelial cellNitric Oxide Synthase.
 9. The method according to any one of claims 1-5,further comprising co-administering an agent other than an agent thatdisrupts actin cytoskeletal organization that increases endothelial cellNitric Oxide Synthase activity.
 10. The method according to any one ofclaims 1-5, further comprising co-administering at least one differentagent that disrupts actin cytoskeletal organization in an amounteffective to increase endothelial cell Nitric Oxide Synthase activity insaid tissue of the subject.